Methods and compositions for targeted polynucleotide modification

ABSTRACT

A variety of methods and compostions are provided, including methods and compositions for targeted modification of a specific target site in a cell or organism, methods for integrating polynucleotides of interest, methods to assess promoter activity, directly select transformed organisms, minimize or eliminate expression resulting from random integration into the genome of an organism, such as a plant, remove polynucleotides of interest, combine multiple transfer cassettes, invert or excise a polynucleotide, silence a gene, and identify and/or characterize transcriptional regulating regions. The methods involve the introduction of a cell proliferation factor and a double-strand break-inducing enzyme into an organism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/291,207, filed on Dec. 30, 2009, the contents of which are hereby incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 399825SEQLIST.TXT, created on Dec. 29, 2010, and having a size of 431 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology, specifically the targeted modification of polynucleotides, including targeted mutagenesis and recombination events.

BACKGROUND OF THE INVENTION

Random insertion of introduced DNA into the genome of a host cell can be lethal if the foreign DNA disrupts an important native gene or regulatory region. Even if a random insertion event does not impair the functioning of a sequence in a host cell, the expression of an inserted foreign nucleotide sequence may be influenced by position effects caused by the surrounding genomic DNA. In some cases, the nucleotide sequence is inserted into a site where the position effect suppresses the function or regulation of the introduced nucleotide sequence. In other instances, overproduction of the gene product may have deleterious effects on a cell.

For example, in plants, position effects can result in reduced agronomics, additional costs for further research, creation of additional transgenic events, slowing product development. For these reasons, efficient methods are needed to target the insertion of nucleotide sequences into the genome of various organisms, such as plants, at chromosomal positions that allow for the desired function of the sequence of interest.

BRIEF SUMMARY OF THE INVENTION

Methods and compositions for targeted modification of a specific target site in a cell are provided. A variety of compositions and methods that can be used to modify a target site are provided, including methods to recombine polynucleotides, assess promoter activity, directly select transformed organisms, minimize or eliminate expression resulting from random integration into the genome of an organism, such as a plant, remove polynucleotides of interest, combine multiple transfer cassettes, invert or excise a polynucleotide, silence gene(s), and characterize transcriptional regulatory regions. The methods involve the introduction of a cell proliferation factor and a double-strand break-inducing enzyme into an organism, and in some embodiments, the introduction of a transfer cassette. Compositions also include plant cells and plants comprising a heterologous polynucleotide encoding a cell proliferation factor, a double-strand break-inducing enzyme and a transfer cassette comprising a recognition sequence that is recognized by the double-strand break-inducing enzyme.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a depiction of a phylogenetic analysis of 50 sequences with homology to maize babyboom (BBM).

FIGS. 2A-2M show the consensus motif sequences 1-10, 14, 15, and 19, respectively, discovered in the analysis described herein, along with the alignments of the regions of various polypeptides used to generate the consensus motifs.

FIG. 3 depicts the motifs found within 50 sequences with homology to maize BBM (ZmBBM).

FIG. 4 shows an alignment of the amino acid sequence of various BBM polypeptides: maize babyboom 2 (ZmBBM2; SEQ ID NO: 29), sorghum babyboom 2 (SbBBM2; SEQ ID NO: 41), rice babyboom 2 (OsBBM2; SEQ ID NO: 35), rice babyboom 3 (OsBBM3; SEQ ID NO: 37), rice babyboom 1 (OsBBM1; SEQ ID NO: 33), maize babyboom (ZmBBM; SEQ ID NO: 2), sorghum babyboom (SbBBM; SEQ ID NO: 39), rice babyboom (OsBBM; SEQ ID NO: 31), Brassica babyboom 1 (BnBBM1; SEQ ID NO: 19), Brassica babyboom 2 (BnBBM2; SEQ ID NO: 21), Arabidopsis babyboom (AtBBM; SEQ ID NO: 17), medicago babyboom (MtBBM; SEQ ID NO: 23), soybean babyboom (GmBBM; SEQ ID NO: 25), and grape babyboom (VvBBM; SEQ ID NO: 27).

FIG. 5 provides a depiction of the motifs found in babyboom polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

Various compositions and methods for modifying a target site in a cell, for example a plant cell, are provided. The modification can include a deletion, mutation, replacement or insertion of a nucleotide sequence. The target site is modified through the activity of a double-strand break-inducing enzyme that recognizes a recognition sequence within the target site. The methods further involve the introduction of a cell proliferation factor, such as a babyboom polypeptide and/or a Wuschel polypeptide, that serves to enhance and promote the modification reaction.

Double-strand breaks induced by double-strand inducing enzymes can result in the induction of DNA repair mechanisms, including the non-homologous end joining pathway, and homologous recombination. Error-prone DNA repair mechanisms can produce mutations at double-strand break sites. The nonhomologous end joining (NHEJ) pathways are the most common repair mechanism that serve to bring the broken polynucleotide ends together (Bleuyard et al. (2006) DNA Repair 5:1-12). The structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible. The two ends of one double-strand break are the most prevalent substrates of NHEJ (Kirik et al. (2000) EMBO J. 19:5562-6). If two different double-strand breaks occur, however, the free ends from different breaks can be ligated to one another, resulting in chromosomal deletions (Siebert and Puchta (2002) Plant Cell 14:1121-31), or chromosomal translocations between different chromosomes (Pacher et al. (2007) Genetics 175:21-9).

Episomal DNA molecules, for example T-DNAs, can also be ligated into the double-strand break, resulting in integration of the episomal DNA molecule into the host genome (Chilton and Que (2003) Plant Physiol 133:956-65; Salomon and Puchta (1998) EMBO J 17:6086-95). Once the sequence around the double-strand breaks is altered, for example, by exonuclease activities involved in the maturation of double-strand breaks, gene conversion pathways can restore the original structure if a homologous sequence is available, such as a homologous chromosome in non-dividing somatic cells, or a sister chromatid after DNA replication (S, G2, M phases of a cell cycle) (Molinier et al. (2004) Plant Cell 16:342-52). Ectopic and/or epigenic DNA sequences may also serve as a DNA repair template for homologous recombination (Puchta (1999) Genetics 152:1173-81).

DNA double-strand breaks (DSBs) appear to be an effective factor to stimulate homologous recombination pathways in every organism tested to date (Puchta et al. (1995) Plant Mol Biol 28:281-92; Tzfira and White (2005) Trends Biotechnol 23:567-9; Puchta (2005) J Exp Bot 56:1-14). For example, using DNA break-inducing enzymes, a two- to nine-fold increase of homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al. (1995) Plant Mol Biol 28:281-92). Thus, double-strand break-inducing enzymes can be used for targeted modification of polynucleotides in organisms and the provision of one or more cell proliferation factors enhances the frequency of targeted modification.

Cell proliferation factors can enhance the rate of targeted modification of a target site in a cell of an organism, such as a plant, that has been induced by a double-strand break-inducing enzyme. In these methods, at least one cell proliferation factor and a double-strand break-inducing enzyme are introduced into a cell having a target site with at least one recognition sequence. The double-strand break-inducing enzyme recognizes the recognition sequence and introduces a double-strand break at or near the recognition sequence to produce a modified target site. Modifications to the target site can include a deletion, mutation, replacement, homologous recombination, or insertion of a nucleotide sequence. In certain embodiments, the target site is stably integrated into the genome of the plant. In some of these embodiments, the genomic target site is a native genomic target site. These methods can be used to stimulate recombination at a target site, integrate polynucleotides into a target site, invert or excise a polynucleotide, directly select transformed organisms, minimize or eliminate expression resulting from random integration into the genome of an organism, combine multiple transfer cassettes, silence genes, and characterize transcriptional regulatory regions.

The presently disclosed methods and compositions utilize cell proliferation factors to enhance rates of targeted polynucleotide modification. As used herein, a “cell proliferation factor” is a polypeptide or a polynucleotide capable of stimulating growth of a cell or tissue, including but not limited to promoting progression through the cell cycle, inhibiting cell death, such as apoptosis, stimulating cell division, and/or stimulating embryogenesis. The polynucleotides can fall into several categories, including but not limited to, cell cycle stimulatory polynucleotides, developmental polynucleotides, anti-apoptosis polynucleotides, hormone polynucleotides, or silencing constructs targeted against cell cycle repressors or pro-apoptotic factors. The following are provided as non-limiting examples of each category and are not considered a complete list of useful polynucleotides for each category: 1) cell cycle stimulatory polynucleotides including plant viral replicase genes such as RepA, cyclins, E2F, prolifera, cdc2 and cdc25; 2) developmental polynucleotides such as Lec1, Kn1 family, WUSCHEL, Zwille, BBM, Aintegumenta (ANT), FUS3, and members of the Knotted family, such as Kn1, STM, OSH1, and SbH1; 3) anti-apoptosis polynucleotides such as CED9, Bcl2, Bcl-X(L), Bcl-W, A1, McL-1, Mac1, Boo, and Bax-inhibitors; 4) hormone polynucleotides such as IPT, TZS, and CKI-1; and 5) silencing constructs targeted against cell cycle repressors, such as Rb, CK1, prohibitin, and wee1, or stimulators of apoptosis such as APAF-1, bad, bax, CED-4, and caspase-3, and repressors of plant developmental transitions, such as Pickle and WD polycomb genes including FIE and Medea. The polynucleotides can be silenced by any known method such as antisense, RNA interference, cosuppression, chimerplasty, or transposon insertion.

The cell proliferation factors can be introduced into cells to enhance targeted polynucleotide modification through the introduction of a polynucleotide that encodes the proliferation factor. The use of the term “polynucleotide” is not intended to limit the compositions to polynucleotides comprising DNA. Polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides also encompass all forms of sequences including, but not limited to, single-, double-, or multi-stranded forms, hairpins, stem-and-loop structures, circular plasmids, and the like. The polynucleotide encoding the cell proliferation factor may be native to the cell or heterologous. A native polypeptide or polynucleotide comprises a naturally occurring amino acid sequence or nucleotide sequence. “Heterologous” in reference to a polypeptide or a nucleotide sequence is a polypeptide or a sequence that originates from a different species, or if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.

Any of a number of cell proliferation factors can be used. In certain embodiments, those cell proliferation factors that are capable of stimulating embryogenesis are used to enhance targeted polynucleotide modification. Such cell proliferation factors are referred to herein as embryogenesis-stimulating polypeptides and they include, but are not limited to, babyboom polypeptides.

In some embodiments, the cell proliferation factor is a member of the AP2/ERF family of proteins. The AP2/ERF family of proteins is a plant-specific class of putative transcription factors that regulate a wide variety of developmental processes and are characterized by the presence of an AP2 DNA binding domain that is predicted to form an amphipathic alpha helix that binds DNA (PFAM Accession PF00847). The AP2 domain was first identified in APETALA2, an Arabidopsis protein that regulates meristem identity, floral organ specification, seed coat development, and floral homeotic gene expression. The AP2/ERF proteins have been subdivided into distinct subfamilies based on the presence of conserved domains. Initially, the family was divided into two subfamilies based on the number of DNA binding domains, with the ERF subfamily having one DNA binding domain, and the AP2 subfamily having 2 DNA binding domains. As more sequences were identified, the family was subsequently subdivided into five subfamilies: AP2, DREB, ERF, RAV, and others. (Sakuma et al. (2002) Biochem Biophys Res Comm 290:998-1009).

Members of the APETALA2 (AP2) family of proteins function in a variety of biological events, including but not limited to, development, plant regeneration, cell division, embryogenesis, and cell proliferation (see, e.g., Riechmann and Meyerowitz (1998) Biol Chem 379:633-646; Saleh and Pages (2003) Genetika 35:37-50 and Database of Arabidopsis Transcription Factors at daft.cbi.pku.edu.cn). The AP2 family includes, but is not limited to, AP2, ANT, Glossy15, AtBBM, BnBBM, and maize ODP2/BBM.

Provided herein is an analysis of fifty sequences with homology to a maize BBM sequence (also referred to as maize ODP2 or ZmODP2, the polynucleotide and amino acid sequence of the maize BBM is set forth in SEQ ID NO: 1 and 2, respectively; the polynucleotide and amino acid sequence of another ZmBBM is set forth in SEQ ID NO: 121 and 122, respectively; and genomic sequences of ZmBBM are set forth in SEQ ID NO: 59 and 101). The analysis identified three motifs (motifs 4-6; set forth in SEQ ID NOs: 6-8), along with the AP2 domains (motifs 2 and 3; SEQ ID NOs: 4 and 5) and linker sequence that bridges the AP2 domains (motif 1; SEQ ID NO: 3), that are found in all of the BBM homologues. Thus, motifs 1-6 distinguish these BBM homologues from other AP2-domain containing proteins (e.g., WRI, AP2, and RAP2.7). Thus, these BBM homologues comprise a subgroup of AP2 family of proteins referred to herein as the BBM/PLT subgroup. In some embodiments, the cell proliferation factor that is used in the methods and compositions is a member of the BBM/PLT group of AP2 domain-containing polypeptides. In these embodiments, the cell proliferation factor comprises two AP2 domains and motifs 4-6 (SEQ ID NOs: 6-8) or a fragment or variant thereof. In some of these embodiments, the AP2 domains have the sequence set forth in SEQ ID NOs: 4 and 5 or a fragment or variant thereof, and in particular embodiments, further comprises the linker sequence of SEQ ID NO: 3 or a fragment or variant thereof. In other embodiments, the cell proliferation factor comprises at least one of motifs 4-6 or a fragment or variant thereof, along with two AP2 domains, which in some embodiments have the sequence set forth in SEQ ID NO: 4 and/or 5 or a fragment or variant thereof, and in particular embodiments have the linker sequence of SEQ ID NO: 3 or a fragment or variant thereof. Based on the phylogenetic analysis provided herein, the subgroup of BBM/PLT polypeptides can be subdivided into the BBM, AIL6/7, PLT1/2, AIL1, PLT3, and ANT groups of polypeptides.

In some embodiments, the cell proliferation factor is a babyboom (BBM) polypeptide, which is a member of the AP2 family of transcription factors. The BBM protein from Arabidopsis (AtBBM) is preferentially expressed in the developing embryo and seeds and has been shown to play a central role in regulating embryo-specific pathways. Overexpression of AtBBM has been shown to induce spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. See, Boutiler et al. (2002) The Plant Cell 14:1737-1749. The maize BBM protein also induces embryogenesis and promotes transformation (See, U.S. Pat. No. 7,579,529, which is herein incorporated by reference in its entirety). Thus, BBM polypeptides stimulate proliferation, induce embryogenesis, enhance the regenerative capacity of a plant, enhance transformation, and as demonstrated herein, enhance rates of targeted polynucleotide modification. As used herein “regeneration” refers to a morphogenic response that results in the production of new tissues, organs, embryos, whole plants or parts of whole plants that are derived from a single cell or a group of cells. Regeneration may proceed indirectly via a callus phase or directly, without an intervening callus phase. “Regenerative capacity” refers to the ability of a plant cell to undergo regeneration.

In some embodiments, the babyboom polypeptide comprises two AP2 domains and at least one of motifs 7 and 10 (set forth in SEQ ID NO: 9 and 12, respectively) or a variant or fragment thereof. In certain embodiments, the AP2 domains are motifs 3 and 2 (SEQ ID NOs: 5 and 4, respectively) or a fragment or variant thereof, and in particular embodiments, the babyboom polypeptide further comprises a linker sequence between AP2 domain 1 and 2 having motif 1 (SEQ ID NO: 3) or a fragment or variant thereof. In particular embodiments, the BBM polypeptide further comprises motifs 4-6 (SEQ ID NOs 6-8) or a fragment or variant thereof. The BBM polypeptide can further comprise motifs 8 and 9 (SEQ ID NOs: 10 and 11, respectively) or a fragment or variant thereof, and in some embodiments, motif 10 (SEQ ID NO: 12) or a variant or fragment thereof. In some of these embodiments, the BBM polypeptide also comprises at least one of motif 14 (set forth in SEQ ID NO: 13), motif 15 (set forth in SEQ ID NO: 14), and motif 19 (set forth in SEQ ID NO: 15), or variants or fragments thereof. The variant of a particular amino acid motif can be an amino acid sequence having at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity with the motif disclosed herein. Alternatively, variants of a particular amino acid motif can be an amino acid sequence that differs from the amino acid motif by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

Non-limiting examples of babyboom polynucleotides or polypeptides that can be used in the methods and compositions include the Arabidopsis thaliana AtBBM (SEQ ID NOs: 16 and 17), Brassica napus BnBBM1 (SEQ ID NOs: 18 and 19), Brassica napus BnBBM2 (SEQ ID NOs: 20 and 21), Medicago truncatula MtBBM (SEQ ID NOs: 22 and 23), Glycine max GmBBM (SEQ ID NOs: 24 and 25), Vitis vinifera VvBBM (SEQ ID NOs: 26 and 27), Zea mays ZmBBM (SEQ ID NOs: 1 and 2 and genomic sequence set forth in SEQ ID NO: 59; and SEQ ID NOs: 104 and 105 and genomic sequence set forth in SEQ ID NO: 101) and ZmBBM2 (SEQ ID NOs: 28 and 29), Oryza sativa OsBBM (polynucleotide sequences set forth in SEQ ID NOs: 30 and 103 and amino acid sequence set forth in SEQ ID NO: 31; genomic sequence set forth in SEQ ID NO: 102), OsBBM1 (SEQ ID NOs: 32 and 33), OsBBM2 (SEQ ID NOs: 34 and 35), and OsBBM3 (SEQ ID NOs: 36 and 37), Sorghum bicolor SbBBM (SEQ ID NOs: 38 and 39 and genomic sequence set forth in SEQ ID NO: 60) and SbBBM2 (SEQ ID NOs: 40 and 41) or active fragments or variants thereof. In particular embodiments, the cell proliferation factor is a maize BBM polypeptide (SEQ ID NO: 2, 29, or 105) or a variant or fragment thereof, or is encoded by a maize BBM polynucleotide (SEQ ID NO: 1, 28, or 104) or a variant or fragment thereof.

In some embodiments, a polynucleotide encoding a cell proliferation factor has a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 59, 101, 102, 103, 104, or 60 or the cell proliferation factor has an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 105, or 41. In some of these embodiments, the cell proliferation factor has at least one of motifs 7 and 10 (SEQ ID NO: 9 and 12, respectively) or a variant or fragment thereof at the corresponding amino acid residue positions in the babyboom polypeptide. In other embodiments, the cell proliferation factor further comprises at least one of motif 14 (set forth in SEQ ID NO: 13), motif 15 (set forth in SEQ ID NO: 14), and motif 19 (set forth in SEQ ID NO: 15) or a variant or fragment thereof at the corresponding amino acid residue positions in the babyboom polypeptide.

In other embodiments, other cell proliferation factors, such as, Lec1, Kn1 family, WUSCHEL (e.g., WUS1, the polynucleotide and amino acid sequence of which is set forth in SEQ ID NO: 51 and 52; WUS2, the polynucleotide and amino acid sequence of which is set forth in SEQ ID NO: 57 and 58; WUS2 alt, the polynucleotide and amino acid sequence of which is set forth in SEQ ID NO: 99 and 100; WUS3, the polynucleotide and amino acid sequence of which is set forth in SEQ ID NO: 97 and 98), Zwille, and Aintegumeta (ANT), may be used alone, or in combination with a babyboom polypeptide or other cell proliferation factor to enhance targeted polynucleotide modification in plants. See, for example, U.S. Application Publication No. 2003/0135889, International Application Publication No. WO 03/001902, and U.S. Pat. No. 6,512,165, each of which is herein incorporated by reference. When multiple cell proliferation factors are used, or when a babyboom polypeptide is used along with any of the abovementioned polypeptides, the polynucleotides encoding each of the factors can be present on the same expression cassette or on separate expression cassettes. Likewise, the polynucleotide(s) encoding the cell proliferation factor(s) and the polynucleotide encoding the double-strand break-inducing enzyme can be located on the same or different expression cassettes. When two or more factors are coded for by separate expression cassettes, the expression cassettes can be provided to the plant simultaneously or sequentially.

In some embodiments, polynucleotides or polypeptides having homology to a known babyboom polynucleotide or polypeptide and/or sharing conserved functional domains can be identified by screening sequence databases using programs such as BLAST. The databases can be queried using full length sequences, or with fragments including, but not limited to, conserved domains or motifs. In some embodiments, the sequences retrieved from the search can be further characterized by alignment programs to quickly identify and compare conserved functional domains, regions of highest homology, and nucleotide and/or amino differences between sequences, including insertions, deletions, or substitutions, including those programs described in more detail elsewhere herein. The retrieved sequences can also be evaluated using a computer program to analyze and output the phylogenetic relationship between the sequences.

In other embodiments, polynucleotides or polypeptides having homology to a known babyboom polynucleotide or polypeptide and/or sharing conserved functional domains can be identified using standard nucleic acid hybridization techniques, such as those described in more detail elsewhere herein. Extensive guides on nucleic acid hybridization include Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, NY); Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, NY); and, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

According to the presently disclosed methods, cell proliferation factors are introduced into cells to enhance the modification of a target site within the cell. The terms “target site,” and “target sequence,” as used interchangeably herein, refer to a polynucleotide sequence present in a cell of an organism, such as a plant, that comprises at least one recognition sequence and/or a nick/cleavage site for a double-strand break-inducing enzyme. The target site may be part of the organism's native genome or integrated therein or may be present on an episomal polynucleotide. The genomic target sequence may be on any region of any chromosome, and may or may not be in a region encoding a protein or RNA. The target site may be native to the cell or heterologous. In some embodiments, the heterologous target sequence may have been transgenically inserted into the organism's genome, and may be on any region of any chromosome, including an artificial or satellite chromosome, and may or may not be in a region encoding a protein or RNA. It is recognized that the cell or the organism may comprise multiple target sites, which may be located at one or multiple loci within or across chromosomes. Multiple independent manipulations of each target site in the organism can be performed using the presently disclosed methods.

The target sites comprise at least one recognition sequence. As used herein, the terms “recognition sequence” or “recognition site,” used interchangeably herein, refer to any nucleotide sequence that is specifically recognized and/or bound by a double-strand break-inducing enzyme. The length of the recognition site sequence can vary, and includes, for example, sequences that are at least about 3, 4, 6, 8, 10, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 80, 90, 100, or more nucleotides in length. In some embodiments, the recognition site is of a sufficient length to only be present in a genome of an organism one time. In some embodiments, the recognition site is palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary strand. The double-strand break-inducing enzyme recognizes the recognition sequence and introduces a double-strand break at or near the recognition sequence. The nick/cleavage site could be within the sequence that is specifically recognized by the enzyme or the nick/cleavage site could be outside of the sequence that is specifically recognized by the enzyme. In some embodiments, the double-strand break is introduced about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more nucleotides away from the recognition sequence.

In some embodiments, the cleavage occurs at nucleotide positions immediately opposite each other to produce a blunt end cut or, in alternative embodiments, the cuts are staggered to produce single-stranded overhangs, also called “sticky ends”, which can be either 5′ overhangs, or 3′ overhangs. The recognition sequence can be endogenous (native) or heterologous to the plant cell. When the recognition site is an endogenous sequence, it may be recognized by a naturally-occurring, or native double-strand break-inducing enzyme. Alternatively, an endogenous recognition sequence may be recognized and/or bound by a modified or engineered double-strand break-inducing enzyme designed or selected to specifically recognize the endogenous recognition sequence to produce a double-strand break.

A double-strand break-inducing enzyme is any enzyme that recognizes and/or binds to a specific recognition sequence to produce a double-strand break at or near the recognition sequence. The double-strand break could be due to the enzymatic activity of the enzyme itself or the enzyme might introduce a single-stranded nick in the DNA that then leads to a double-strand break induced by other cellular machinery (e.g., cellular repair mechanisms). Examples of double-strand break-inducing enzymes include, but are not limited to, endonucleases, site-specific recombinases, transposases, topoisomerases, and zinc finger nucleases, and include modified derivatives, variants, and fragments thereof. A modified double-strand break-inducing enzyme can be derived from a native, naturally-occurring double-strand break-inducing enzyme or it can be artificially created or synthesized. Those modified double-strand break-inducing enzymes that are derived from a native, naturally-occurring double-strand break-inducing enzymes can be modified to recognize a different recognition sequence (at least one nucleotide difference) than its native form. In certain embodiments, the double-strand break-inducing enzyme recognizes recognition sequences that are of a sufficient length to have only one copy in a genome of an organism.

In some embodiments, the double-strand break-inducing enzyme can be provided to an organism through the introduction of a polynucleotide encoding the enzyme. In some of these embodiments, the polynucleotide can be modified to at least partially optimize codon usage in the organism, such as plants. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, WO 99/25841, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Such polynucleotides wherein the frequency of codon usage has been designed to mimic the frequency of preferred codon usage of the host cell are referred to herein as being “codon-modified”, “codon-preferred”, or “codon-optimized.” The polynucleotide encoding the cell proliferation factor, and in some embodiments, the polynucleotide of interest, can also be at least partially modified to optimized codon usage in the host cell or organism.

In some embodiments, the double-strand break-inducing enzyme is a transposase. Transposases are polypeptides that mediate transposition of a transposon from one location in the genome to another. Transposases typically induce double-strand breaks to excise the transposon, recognize subterminal repeats, and bring together the ends of the excised transposon, in some systems, other proteins are also required to bring together the ends during transposition. Examples of transposons and transposases include, but are not limited to, the Ac/Ds, Dt/rdt, Mu-M1/Mn, and Spm(En)/dSpm elements from maize, the Tam elements from snapdragon, the Mu transposon from bacteriophage, bacterial transposons (Tn) and insertion sequences (IS), Ty elements of yeast (retrotransposon), Ta1 elements from Arabidopsis (retrotransposon), the P element transposon from Drosophila (Gloor et al. (1991) Science 253:1110-1117), the Copia, Mariner and Minos elements from Drosophila, the Hermes elements from the housefly, the PiggyBack elements from Trichplusia ni, Tc1 elements from C. elegans, and IAP elements from mice (retrotransposon).

In other embodiments, the double-strand break-inducing enzyme is a DNA topoisomerase. DNA topoisomerases modulate DNA secondary and higher order structures and functions related primarily to replication, transcription, recombination and repair. Topoisomerases share two characteristics: (i) the ability to cleave and reseal the phosphodiester backbone of DNA in two successive transesterification reactions; and (ii) once a topoisomerase cleaved DNA intermediate is formed, the enzyme allows the severed DNA ends to come apart, allowing the passage of another single- or double-stranded DNA segment. DNA topoisomerases can be classified into three evolutionary independent families: type IA, type IB and type II.

Type IA and type IB topoisomerases cleave only a single strand of DNA. The Escherichia coli topoisomerase I and topoisomerase III, Saccharomyces cerevisiae topoisomerase III and reverse gyrase belong to the type IA or type I-5′ subfamily as the protein link is to a 5′ phosphate in the DNA. The prototype of type IB or I-3′ enzymes are found in all eukaryotes and also in vaccinia virus topoisomerase I where the protein is attached to a 3′ phosphate. Despite differences in mechanism and specificity between the bacterial and eukaryotic enzymes, yeast DNA topoisomerase I can complement a bacterial DNA topoisomerase I mutant (Bjornsti et al. (1987) Proc Natl Acad Sci USA 84:8971-5). Type IA topoisomerases relax negatively supercoiled DNA and require magnesium and a single-stranded region of DNA. Topoisomerases IB relax both positively and negatively supercoiled DNA with equal efficiency and do not require a single-stranded region of DNA or metal ions for function.

The type II family of DNA topoisomerases are homodimeric (eukaryotic topoisomerase II) or tetrameric (gyrase) enzymes that cleave both strands of a DNA duplex. Type II topoisomerases include, but are not limited to, E. coli DNA gyrase, E. coli topoisomerase IV (par E), eukaryotic type II topoisomerases, and archaic topoisomerase VI. Preferred cutting sites are known for available topoisomerases.

In particular embodiments, the double-strand break-inducing enzyme is an endonuclease. Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, and include restriction endonucleases that cleave DNA at specific sites without damaging the bases. Restriction endonucleases include Type I, Type II, Type III, and Type IV endonucleases, which further include various subtypes. In the Type I and Type III systems, a single protein complex has both methylase and restriction activities.

Type I and Type III restriction endonucleases recognize specific recognition sequences, but typically cleave at a variable position from the recognition site, which can be hundreds of base pairs away from the recognition site. In Type II systems, the restriction activity is independent of any methylase activity, and typically cleavage occurs at specific sites within or near to the recognition site. Most Type II enzymes cut palindromic sequences, however Type IIa enzymes recognize non-palindromic recognition sites and cleave outside of the recognition site; Type IIb enzymes cut sequences twice with both sites outside of the recognition site; and Type IIs endonucleases recognize an asymmetric recognition site and cleave on one side and at a defined distance of about 1-20 nucleotides from the recognition site.

Type IV restriction enzymes target methylated DNA. Restriction enzymes are further described and classified, for example in the REBASE database (on the world wide web at rebase.neb.com; Roberts et al. (2003) Nucleic Acids Res 31:418-20; Roberts et al. (2003) Nucleic Acids Res 31:1805-12; and Belfort et al. (2002) in Mobile DNA II, pp. 761-783, Eds. Craigie, et al., ASM Press, Washington, D.C, each of which is herein incorporated by reference in its entirety).

Endonucleases that are suitable for use in the presently described methods and compositions include homing endonucleases, which like restriction endonucleases, bind and cut polynucleotides at a specific recognition sequence, however the recognition sequences for homing endonucleases are typically longer, about 18 bp or more. These sequences are predicted to naturally occur infrequently in a genome, typically only one or two sites per genome.

Homing endonucleases, also known as meganucleases, have been classified into four families based on conserved sequence motifs: the LAGLIDADG, GIY-YIG, H—N—H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. Homing endonucleases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for homing endonucleases is similar to the convention for other restriction endonucleases. Homing endonucleases are also characterized by a prefix of F-, I-, or PI- for enzymes encoded by free-standing ORFs, introns, and inteins, respectively. For example, the intron-, intein-, and freestanding gene-encoded homing endonucleases from Saccharomyces cerevisiae are denoted I-SceI, PI-SceI, and F-SceII (HO endonuclease), respectively. Homing endonuclease domains, structure and function are known (see for example, Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38:199-248; Lucas et al. (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard (1999) Cell Mol Life Sci 55:1304-26; Stoddard (2006) Q Rev Biophys 38:49-95; and Moure et al. (2002) Nat Struct Biol 9:764, each of which is herein incorporated by reference). In some embodiments, a naturally occurring variant, and/or an engineered derivative homing endonuclease is used. The cleavage specificity of a homing endonuclease can be changed by rational design of amino acid substitutions at the DNA binding domain and/or combinatorial assembly and selection of mutated monomers (see, for example, Arnould et al. (2006) J Mol Biol 355:443-58; Ashworth et al. (2006) Nature 441:656-9; Doyon et al. (2006) J Am Chem Soc 128:2477-84; Rosen et al. (2006) Nucleic Acids Res 34:4791-800; and Smith et al. (2006) Nucleic Acids Res 34:e149, each of which is herein incorporated by reference). Engineered homing endonucleases have been demonstrated that can cleave cognate mutant sites without broadening their specificity. The endonuclease can be a modified endonuclease that binds a non-native or heterologous recognition sequence and does not bind a native or endogenous recognition sequence. An engineered or modified endonuclease can have only a single modified amino acid or many amino acid changes. Methods for modifying the kinetics, cofactor interactions, expression, optimal conditions, and/or recognition site specificity of homing endonucleases, and subsequently screening for activity are known, see for example, Epinat et al. (2003) Nucleic Acids Res 31:2952-62; Chevalier et al. (2002) Mol Cell 10:895-905; Gimble et al. (2003) Mol Biol 334:993-1008; Seligman et al. (2002) Nucleic Acids Res 30:3870-9; Sussman et al. (2004) J Mol Biol 342:31-41; Rosen et al. (2006) Nucleic Acids Res 34:4791-800; Chames et al. (2005) Nucleic Acids Res 33:e178; Smith et al. (2006) Nucleic Acids Res 34:e149; Gruen et al. (2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:e154; U.S. Application Publication No. US2007/0117128; and International Application Publication Nos. WO 05/105989, WO 03/078619, WO 06/097854, WO 06/097853, WO 06/097784, WO 04/031346, WO 04/067753, and WO 07/047,859, each of which is herein incorporated by reference in its entirety.

Any homing endonuclease can be used as a double-strand break inducing agent including, but not limited to, I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NclIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp68031, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-Mtu1, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliII, or any variant or derivative thereof.

In still other embodiments, the double-strand break-inducing enzyme is a zinc finger nuclease. Zinc finger nucleases (ZFNs) are engineered double-strand break inducing agents comprised of a zinc finger DNA binding domain and a double strand break-inducing enzymatic domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprises two, three, four, or more zinc fingers, for example having a C2H2 structure; however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable to the design of polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs consist of an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example, a nuclease domain from a Type IIs endonuclease such as FokI. Additional functionalities can be fused to the zinc-finger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases. In some examples, dimerization of the nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3-finger domain recognizes a sequence of nine contiguous nucleotides, with a dimerization requirement of the nuclease. Two sets of zinc finger triplets are used to bind an 18-nucleotide recognition sequence. A recognition sequence of 18 nucleotides is long enough to be unique in a genome (4¹⁸=6.9×10¹⁰).

To date, designer zinc finger modules predominantly recognize GNN and ANN triplets (Dreier et al. (2001) J Biol Chem 276:29466-78; Dreier et al. (2000) J Mol Biol 303:489-502; Liu et al. (2002) J Biol Chem 277:3850-6, each of which is herein incorporated by reference), but examples using CNN or TNN triplets are also known (Dreier et al. (2005) J Biol Chem 280:35588-97; Jamieson et al. (2003) Nature Rev Drug Discov 2:361-8). See also, Durai et al. (2005) Nucleic Acids Res 33:5978-90; Segal (2002) Methods 26:76-83; Porteus and Carroll (2005) Nat Biotechnol 23:967-73; Pabo et al. (2001) Ann Rev Biochem 70:313-40; Wolfe et al. (2000) Ann Rev Biophys Biomol Struct 29:183-212; Segal and Barbas (2001) Curr Opin Biotechnol 12:632-7; Segal et al. (2003) Biochemistry 42:2137-48; Beerli and Barbas (2002) Nat Biotechnol 20:135-41; Mani et al. (2005) Biochem Biophys Res Comm 335:447-57; Lloyd et al. (2005) Proc Natl Acad Sci USA 102:2232-7; Carroll et al. (2006) Nature Protocols 1:1329; Ordiz et al. (2002) Proc Natl Acad Sci USA 99:13290-5; Guan et al. (2002) Proc Natl Acad Sci USA 99:13296-301; Townsend et al. (2009) Nature 459:442-445; Sander et al. (2008) Nucl Acids Res 37:509-515; Fu et al. (2009) Nucl Acids Res 37:D297-283; Maeder et al. (2008) Mol Cell 31:294-301; Wright et al. (2005) Plant J 44:693-705; Wright et al. (2006) Nat Prot 1:1637-1652; zinc-finger consortium (website at www-dot-zincfinger-dot-org); International Application Publication Nos. WO 02/099084; WO 00/42219; WO 02/42459; WO 03/062455; U.S. Application Publication Nos. 2003/0059767 and 2003/0108880; and U.S. Pat. Nos. 6,534,261, 7,262,054, 7,378,510, 7,151,201, 6,140,466, 6,511,808 and 6,453,242; each of which is herein incorporated by reference in its entirety.

Alternatively, engineered zinc finger DNA binding domains can be fused to other double-strand break-inducing enzymes or derivatives thereof that retain DNA nicking/cleaving activity. For example, this type of fusion can be used to direct the double-strand break-inducing enzyme to a different recognition site, to alter the location of the nick or cleavage site, to direct the inducing agent to a shorter recognition site, or to direct the inducing agent to a longer recognition site. In some embodiments, a zinc finger DNA binding domain is fused to a site-specific recombinase, transposase, topoisomerase, endonuclease, or a derivative thereof that retains DNA nicking and/or cleaving activity.

In some embodiments, a site-specific recombinase is used as the double-strand break-inducing enzyme. A site-specific recombinase, also referred to herein as a recombinase, is a polypeptide that catalyzes conservative site-specific recombination between its compatible recombination sites, and includes native polypeptides as well as derivatives, variants and/or fragments that retain activity, and native polynucleotides, derivatives, variants, and/or fragments that encode a recombinase that retains activity. The recombinase used in the methods and compositions can be a native recombinase or a biologically active fragment or variant of the recombinase. In some embodiments, the site-specific recombinase is a recombinantly produced enzyme or variant thereof, which catalyzes conservative site-specific recombination between specified DNA recombination sites. For reviews of site-specific recombinases and their recognition sites, see Sauer (1994) Curr Op Biotechnol 5:521-527; and Sadowski (1993) FASEB 7:760-767, each of which is herein incorporated by reference in its entirety.

Any recombinase system can be used in the methods and compositions. A recombinase can be provided via a polynucleotide that encodes the recombinase, a modified polynucleotide encoding the recombinase, or the polypeptide itself. Non-limiting examples of site-specific recombinases that can be used to produce a double-strand break at a recognition sequence include FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, U153, and other site-specific recombinases known in the art, including those described in Thomson and Ow (2006) Genesis 44:465-476, which is herein incorporated by reference in its entirety. Examples of site-specific recombination systems used in plants can be found in U.S. Pat. Nos. 5,929,301, 6,175,056, 6,331,661; and International Application Publication Nos. WO 99/25821, WO 99/25855, WO 99/25841, and WO 99/25840, the contents of each are herein incorporated by reference.

In some embodiments, recombinases from the Integrase or Resolvase families are used, including biologically active variants and fragments thereof. The Integrase family of recombinases has over one hundred members and includes, for example, FLP, Cre, lambda integrase, and R. The Integrase family has been grouped into two classes based on the structure of the active sites, serine recombinases and tyrosine recombinases. The tyrosine family, which includes Cre, FLP, SSV1, and lambda integrase, uses the catalytic tyrosine's hydroxyl group for a nucleophilic attack on the phosphodiester bond of the DNA. Typically, members of the tyrosine family initially nick the DNA, which later forms a double strand break. In the serine recombinase family, which includes phiC31 integrase, a conserved serine residue forms a covalent link to the DNA target site (Grindley et al. (2006) Ann Rev Biochem 16:16). For other members of the Integrase family, see, for example, Esposito et al. (1997) Nucleic Acids Res 25:3605-3614; and Abremski et al. (1992) Protein Eng 5:87-91; each of which are herein incorporated by reference in its entirety. Other recombination systems include, for example, the Streptomycete bacteriophage phi C31 (Kuhstoss et al. (1991) J Mol Biol 20:897-908); the SSV1 site-specific recombination system from Sulfolobus shibatae (Maskhelishvili et al. (1993) Mol Gen Genet 237:334-342); and a retroviral integrase-based integration system (Tanaka et al. (1998) Gene 17:67-76). In some embodiments, the recombinase does not require cofactors or a supercoiled substrate. Such recombinases include Cre, FLP, or active variants or fragments thereof.

The FLP recombinase is a protein that catalyzes a site-specific reaction that is involved in amplifying the copy number of the two-micron plasmid of S. cerevisiae during DNA replication. FLP recombinase catalyzes site-specific recombination between two FRT sites. The FLP protein has been cloned and expressed (Cox (1993) Proc Natl Acad Sci USA 80:4223-4227). The FLP recombinase for use in the methods and compositions may be derived from the genus Saccharomyces. In some embodiments, a recombinase polynucleotide modified to comprise more plant-preferred codons is used. A recombinant FLP enzyme encoded by a nucleotide sequence comprising maize preferred codons (FLPm) that catalyzes site-specific recombination events is known (the polynucleotide and polypeptide sequence of which is set forth in SEQ ID NO: 42 and 43, respectively; see, e.g., U.S. Pat. No. 5,929,301, which is herein incorporated by reference in its entirety). Thus, in some embodiments, the site-specific recombinase used in the methods and compositions has the sequence set forth in SEQ ID NO: 43 (FLP) has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 43. In some of those embodiments wherein the site-specific recombinase is provided to the cell through the introduction of a polynucleotide that encodes the site-specific recombinase, the polynucleotide has the sequence set forth in SEQ ID NO: 42 (FLPm) or has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 42. Additional functional variants and fragments of FLP are known (Buchholz et al. (1998) Nat Biotechnol 16:657-662; Hartung et al. (1998) J Biol Chem 273:22884-22891; Saxena et al. (1997) Biochim Biophys Acta 1340:187-204; Hartley et al. (1980) Nature 286:860-864; Voziyanov et al. (2002) Nucleic Acids Res 30:1656-1663; Zhu & Sadowski (1995) J Biol Chem 270:23044-23054; and U.S. Pat. No. 7,238,854, each of which is herein incorporated by reference in its entirety).

The bacteriophage recombinase Cre catalyzes site-specific recombination between two lox sites. The Cre recombinase is known (Guo et al. (1997) Nature 389:40-46; Abremski et al. (1984) J Biol Chem 259:1509-1514; Chen et al. (1996) Somat Cell Mol Genet 22:477-488; Shaikh et al. (1977) J Biol Chem 272:5695-5702; and, Buchholz et al. (1998) Nat Biotechnol 16:657-662, each of which is herein incorporated by reference in its entirety). Cre polynucleotide sequences may also be synthesized using plant-preferred codons, for example such sequences (maize optimized Cre (moCre); the polynucleotide and polypeptide sequence of which is set forth in SEQ ID NO: 44 and 45, respectively) are described, for example, in International Application Publication No. WO 99/25840, which is herein incorporated by reference in its entirety. Thus, in some embodiments, the site-specific recombinase used in the methods and compositions has the sequence set forth in SEQ ID NO: 45 (Cre) has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 45. In some of those embodiments wherein the site-specific recombinase is provided to the cell through the introduction of a polynucleotide that encodes the site-specific recombinase, the polynucleotide has the sequence set forth in SEQ ID NO: 44 (moCre) or has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 44. Variants of the Cre recombinase are known (see, for example U.S. Pat. No. 6,890,726; Rufer & Sauer (2002) Nucleic Acids Res 30:2764-2772; Wierzbicki et al. (1987) J Mol Biol 195:785-794; Petyuk et al. (2004) J Biol Chem 279:37040-37048; Hartung & Kisters-Woike (1998) J Biol Chem 273:22884-22891; Santoro & Schultz (2002) Proc Natl Acad Sci USA 99:4185-4190; Koresawa et al. (2000) J Biochem (Tokyo) 127:367-372; and Vergunst et al. (2000) Science 290:979-982, each of which are herein incorporated by reference in its entirety).

In some embodiments, a chimeric recombinase is used. A chimeric recombinase is a recombinant fusion protein which is capable of catalyzing site-specific recombination between recombination sites that originate from different recombination systems. For example, if the set of recombination sites comprises a FRT site and a LoxP site, a chimeric FLP/Cre recombinase or active variant or fragment thereof can be used, or both recombinases may be separately provided. Methods for the production and use of such chimeric recombinases or active variants or fragments thereof are described, for example, in International Application Publication No. WO 99/25840; and Shaikh & Sadowski (2000) J Mol Biol 302:27-48, each of which are herein incorporated by reference in its entirety.

In other embodiments, a variant recombinase is used. Methods for modifying the kinetics, cofactor interaction and requirements, expression, optimal conditions, and/or recognition site specificity, and screening for activity of recombinases and variants are known, see for example Miller et al. (1980) Cell 20:721-9; Lange-Gustafson and Nash (1984) J Biol Chem 259:12724-32; Christ et al. (1998) J Mol Biol 288:825-36; Lorbach et al. (2000) J Mol Biol 296:1175-81; Vergunst et al. (2000) Science 290:979-82; Dorgai et al. (1995) J Mol Biol 252:178-88; Dorgai et al. (1998) J Mol Biol 277:1059-70; Yagu et al. (1995) J Mol Biol 252:163-7; Sclimente et al. (2001) Nucleic Acids Res 29:5044-51; Santoro and Schultze (2002) Proc Natl Acad Sci USA 99:4185-90; Buchholz and Stewart (2001) Nat Biotechnol 19:1047-52; Voziyanov et al. (2002) Nucleic Acids Res 30:1656-63; Voziyanov et al. (2003) J Mol Biol 326:65-76; Klippel et al. (1988) EMBO J 7:3983-9; Arnold et al. (1999) EMBO J 18:1407-14; and International Application Publication Nos. WO 03/08045, WO 99/25840, and WO 99/25841; each of which is herein incorporated by reference in its entirety. The recognition sites range from about 30 nucleotide minimal sites to a few hundred nucleotides.

By “recombination site” is intended a polynucleotide (native or synthetic/artificial) that is recognized by the recombinase enzyme of interest. As outlined above, many recombination systems are known in the art and one of skill will recognize the appropriate recombination site to be used with the recombinase of interest.

Non-limiting examples of recombination sites include FRT sites including, for example, the native FRT site (FRT1, SEQ ID NO:46), and various functional variants of FRT, including but not limited to, FRT5 (SEQ ID NO:47), FRT6 (SEQ ID NO:48), FRT7 (SEQ ID NO:49), FRT12 (SEQ ID NO: 53), and FRT87 (SEQ ID NO:50). See, for example, International Application Publication Nos. WO 03/054189, WO 02/00900, and WO 01/23545; and Schlake et al. (1994) Biochemistry 33:12745-12751, each of which is herein incorporated by reference. Recombination sites from the Cre/Lox site-specific recombination system can be used. Such recombination sites include, for example, native LOX sites and various functional variants of LOX.

In some embodiments, the recombination site is a functional variant of a FRT site or functional variant of a LOX site, any combination thereof, or any other combination of recombinogenic or non-recombinogenic recombination sites known. Functional variants include chimeric recombination sites, such as an FRT site fused to a LOX site (see, for example, Luo et al. (2007) Plant Biotech J 5:263-274, which is herein incorporated by reference in its entirety). Functional variants also include minimal sites (FRT and/or LOX alone or in combination). The minimal native FRT recombination site (SEQ ID NO: 46) has been characterized and comprises a series of domains comprising a pair of 11 base pair symmetry elements, which are the FLP binding sites; the 8 base pair core, or spacer, region; and the polypyrimidine tracts. In some embodiments, at least one modified FRT recombination site is used. Modified or variant FRT recombination sites are sites having mutations such as alterations, additions, or deletions in the sequence. The modifications include sequence modification at any position, including but not limited to, a modification in at least one of the 8 base pair spacer domain, a symmetry element, and/or a polypyrimidine tract. FRT variants include minimal sites (see, e.g., Broach et al. (1982) Cell 29:227-234; Senecoff et al. (1985) Proc Natl Acad Sci USA 82:7270-7274; Gronostajski & Sadowski (1985) J Biol Chem 260:12320-12327; Senecoff et al. (1988) J Mol Biol 201:405-421; and International Application Publication No. WO99/25821), and sequence variants (see, for example, Schlake & Bode (1994) Biochemistry 33:12746-12751; Seibler & Bode (1997) Biochemistry 36:1740-1747; Umlauf & Cox (1988) EMBO J 7:1845-1852; Senecoff et al. (1988) J Mol Biol 201:405-421; Voziyanov et al. (2002) Nucleic Acids Res 30:7; International Application Publication Nos. WO 07/011,733, WO 99/25854, WO 99/25840, WO 99/25855, WO 99/25853 and WO 99/25821; and U.S. Pat. Nos. 7,060,499 and 7,476,539; each of which are herein incorporated by reference in its entirety).

An analysis of the recombination activity of variant LOX sites is presented in Lee et al. (1998) Gene 216:55-65 and in U.S. Pat. No. 6,465,254. Also, see for example, Huang et al. (1991) Nucleic Acids Res 19:443-448; Sadowski (1995) In Progress in Nucleic Acid Research and Molecular Biology Vol. 51, pp. 53-91; U.S. Pat. No. 6,465,254; Cox (1989) In Mobile DNA, Berg and Howe (eds) American Society of Microbiology, Washington D.C., pp. 116-670; Dixon et al. (1995) Mol Microbiol 18:449-458; Buchholz et al. (1996) Nucleic Acids Res 24:3118-3119; Kilby et al. (1993) Trends Genet 9:413-421; Rossant & Geagy (1995) Nat Med 1:592-594; Albert et al. (1995) Plant J 7:649-659; Bayley et al. (1992) Plant Mol Biol 18:353-361; Odell et al. (1990) Mol Gen Genet 223:369-378; Dale & Ow (1991) Proc Natl Acad Sci USA 88:10558-10562; Qui et al. (1994) Proc Natl Acad Sci USA 91:1706-1710; Stuurman et al. (1996) Plant Mol Biol 32:901-913; Dale et al. (1990) Gene 91:79-85; and International Application Publication No. WO 01/111058; each of which is herein incorporated by reference in its entirety.

Naturally occurring recombination sites or biologically active variants thereof are of use. Methods to determine if a modified recombination site is recombinogenic are known (see, for example, International Application Publication No. WO 07/011,733, which is herein incorporated by reference in its entirety). Variant recognition sites are known, see for example, Hoess et al. (1986) Nucleic Acids Res 14:2287-300; Albert et al. (1995) Plant J 7:649-59; Thomson et al. (2003) Genesis 36:162-7; Huang et al. (1991) Nucleic Acids Res 19:443-8; Siebler and Bode (1997) Biochemistry 36:1740-7; Schlake and Bode (1994) Biochemistry 33:12746-51; Thygarajan et al. (2001) Mol Cell Biol 21:3926-34; Umlauf and Cox (1988) EMBO J 7:1845-52; Lee and Saito (1998) Gene 216:55-65; International Application Publication Nos. WO 01/23545, WO 99/25851, WO 01/11058, WO 01/07572; and U.S. Pat. No. 5,888,732; each of which is herein incorporated by reference in its entirety.

The recombination sites employed in the methods and compositions can be identical or dissimilar sequences. Recombination sites with dissimilar sequences can be either recombinogenic or non-recombinogenic with respect to one another.

By “recombinogenic” is intended that the set of recombination sites (i.e., dissimilar or corresponding) are capable of recombining with one another. Alternatively, by “non-recombinogenic” is intended the set of recombination sites, in the presence of the appropriate recombinase, will not recombine with one another or recombination between the sites is minimal. Accordingly, it is recognized that any suitable set of non-recombinogenic and/or recombinogenic recombination sites may be utilized, including a FRT site or functional variant thereof, a LOX site or functional variant thereof, any combination thereof, or any other combination of non-recombinogenic and/or recombination sites known in the art.

In some embodiments, the recombination sites are asymmetric, and the orientation of any two sites relative to each other will determine the recombination reaction product. Directly repeated recombination sites are those recombination sites in a set of recombinogenic recombination sites that are arranged in the same orientation, such that recombination between these sites results in excision, rather than inversion, of the intervening DNA sequence. Inverted recombination sites are those recombination sites in a set of recombinogenic recombination sites that are arranged in the opposite orientation, so that recombination between these sites results in inversion, rather than excision, of the intervening DNA sequence.

Fragments and variants of the polynucleotides encoding double-strand break-inducing enzymes and cell proliferation factors and fragments and variants of the double-strand break-inducing enzymes and cell proliferation proteins can be used in the methods and compositions. By “fragment” is intended a portion of the polynucleotide and hence the protein encoded thereby or a portion of the polypeptide. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and hence implement a double-strand break (double-strand break-inducing enzyme) or stimulate cell growth (cell proliferation factor). Thus, fragments of a polynucleotide may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, about 500 nucleotides, about 1000 nucleotides, and up to the full-length polynucleotide encoding a double-strand break-inducing enzyme or cell proliferation factor.

A fragment of a polynucleotide that encodes a biologically active portion of a double-strand break-inducing enzyme or a cell proliferation protein will encode at least about 15, 25, 30, 50, 100, 150, 200, 250, 300, 320, 350, 375, 400, or 500 contiguous amino acids, or up to the total number of amino acids present in a full-length double-strand break-inducing enzyme or cell proliferation protein used in the methods or compositions.

A biologically active portion of a double-strand break-inducing enzyme or cell proliferation protein can be prepared by isolating a portion of one of the polynucleotides encoding the portion of the double-strand break-inducing enzyme or cell proliferation polypeptide and expressing the encoded portion of the double-strand break-inducing enzyme or cell proliferation protein, and assessing the activity of the portion of the double-strand break-inducing enzyme or cell proliferation factor. Polynucleotides that encode fragments of a double-strand break-inducing enzyme or cell proliferation polypeptide can comprise nucleotide sequence comprising at least about 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, or 1,500 nucleotides, or up to the number of nucleotides present in a full-length double-strand break-inducing enzyme or cell proliferation factor nucleotide sequence disclosed herein.

“Variant” sequences have a high degree of sequence similarity. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the native recombinase polypeptides. Variants such as these can be identified with the use of well-known molecular biology techniques, such as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a biologically active protein, such as a double-strand break inducing agent or a cell proliferation factor. Generally, variants of a particular polynucleotide will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by known sequence alignment programs and parameters.

Variants of a particular polynucleotide (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, isolated polynucleotides that encode a polypeptide with a given percent sequence identity to the recombinase are known in the art. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described. Where any given pair of polynucleotides is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

A variant protein can be derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, introduce a double-strand break at or near a recognition sequence (double-strand break-inducing enzyme) or stimulate cell growth (cell proliferation factor). Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native double-strand break-inducing protein or cell proliferation factor will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by known sequence alignment programs and parameters. A biologically active variant of a protein may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The introduction of a cell proliferation factor into a cell can also enhance the rate of targeted integration of a polynucleotide of interest. In these methods, at least one cell proliferation factor is introduced into a cell and a double-strand break-inducing enzyme is introduced, along with a transfer cassette comprising the polynucleotide of interest. As used herein, a “transfer cassette” refers to a polynucleotide that can be introduced into a cell, wherein the polynucleotide comprises a polynucleotide of interest that is to be inserted into a target site of a cell. The introduction of a double-strand break can result in the integration of the polynucleotide of interest through non-homologous end joining or if the transfer cassette comprises at least one region of homology to the target site, the polynucleotide of interest can be integrated through homologous recombination.

Homology indicates at least two sequences that have structural similarity such that they are recognized as being structurally or functionally related sequences. For example, homology indicates that two polynucleotide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction. Homology can be described or identified in by any known means. In some examples, homology is described using percent sequence identity or sequence similarity, for example by using computer implemented algorithms to search or measure the sequence identity and similarity. Sequence identity or similarity may exist over the full length of a sequence, or may be less evenly distributed, for example it may be significantly higher in a conserved domain region.

The amount of homology or sequence identity shared by two sequences can vary and includes total lengths and/or regions having unit integral values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site. These ranges include every integer within the range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bp. The amount of homology can also be described by percent sequence identity over the full aligned length of the two polynucleotides which includes percent sequence identity of about at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus.

Homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, which is described elsewhere herein (see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Current Protocols in Molecular Biology, Ausubel, et al., Eds (1994) Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc; and, Tijssen, (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Elsevier, New York).

In those embodiments wherein the transfer cassette comprises at least one region of homology to a region of the target site, there is sufficient homology between the two regions to allow for homologous recombination to occur between the transfer cassette and the target site. In some embodiments, the transfer cassette comprises a first region of homology to the target site, which can be the recognition sequence, and the polynucleotide of interest. In other embodiments, the transfer cassette comprises a first region of homology to the target site, a polynucleotide of interest, and a second region of homology to the target site. In some of these embodiments, the regions of homology are recombination sites and the double-strand break-inducing enzyme is a site-specific recombinase, such as FLP, Cre, SSVI, R, Int, lambda, phiC31, or HK022. The first and the second recombination site can be recombinogenic or non-recombinogenic with respect to one another. In other embodiments, the region(s) of homology of the transfer cassette to the target site are homologous to other regions of the target site, which can comprise genomic sequence.

In specific embodiments wherein the double-strand break-inducing enzyme that is introduced into a cell along with at least one cell proliferation factor is a site-specific recombinase, the target site of the cell comprises a first recombination site, and a transfer cassette is further introduced into the cell that comprises a second site-specific recombination site and a polynucleotide of interest, wherein the first and the second recombination sites are recombinogenic with each other in the presence of the site-specific recombinase, the polynucleotide of interest can be inserted at the target site. The first and the second recombination sites can be identical or dissimilar.

In other specific embodiments, the introduction of at least one cell proliferation factor into a cell can also enhance the rate of insertion of a polynucleotide of interest into a target site in a cell, wherein the target site comprises a first and a second recombination site that are dissimilar and non-recombinogenic with respect to one another, wherein the recombination sites flank a nucleotide sequence, through the further introduction of a site-specific recombinase, and a transfer cassette comprising a third and a fourth recombination site flanking a polynucleotide of interest, wherein the third recombination site is recombinogenic with the first recombination site, and the fourth recombination site is recombinogenic with the second recombination site in the presence of the site-specific recombinase. The nucleotide sequence between the recombination sites of the target site will be exchanged with the polynucleotide of interest between the recombination sites of the transfer cassette.

As used herein, the term “flanked by”, when used in reference to the position of the recombination sites or regions of homology of the target site or the transfer cassette, refers to a position immediately adjacent to the sequence intended to be exchanged or inserted.

The recombination sites or regions of homology of the transfer cassette may be directly contiguous with the polynucleotide of interest or there may be one or more intervening sequences present between one or both ends of the polynucleotide of interest and the recombination sites or regions of homology. Intervening sequences of particular interest include linkers, adapters, selectable markers, additional polynucleotides of interest, promoters, and/or other sites that aid in vector construction or analysis. It is further recognized that the recombination sites or regions of homology can be contained within the polynucleotide of interest (i.e., such as within introns, coding sequence, or 5′ and 3′ untranslated regions).

A method to directly select a transformed cell or an organism (such as a plant or plant cell) is provided. The method comprises providing a cell or organism having a polynucleotide comprising a target site. The polynucleotide comprises, in the following order, a promoter and a target site. A transfer cassette is introduced into the cell or organism, where the transfer cassette comprises, in the following order, a first region of homology with the target site, a polynucleotide comprising a selectable marker not operably linked to a promoter, and a second region of homology with the target site. At least one cell proliferation factor (e.g., babyboom polypeptide) and a double-strand break-inducing enzyme are introduced into the cell or into the organism and the selectable marker is integrated into the target site. The cell or organism is then grown on the appropriate selective agent to recover the organism that has successfully undergone targeted integration of the selectable marker at the target site. In certain embodiments, the target site is stably integrated into the genome of the plant. In some of these embodiments, the genomic target site is a native genomic target site.

In specific embodiments of the method for directly selecting a transformed cell or an organism as described herein, the cell or the organism has a polynucleotide comprising, in the following order, a promoter and a target site that comprises a first and a second recombination site, wherein the first and the second recombination sites are dissimilar and non-recombinogenic with respect to one another. A transfer cassette is introduced into the cell or organism, wherein the transfer cassette comprises, in the following order, a first recombination site, a polynucleotide comprising a selectable marker not operably linked to a promoter, and a second recombination site, wherein the first and the second recombination sites are non-recombinogenic with respect to one another. A cell proliferation factor and a site-specific recombinase is introduced into the cell or organism and the selectable marker is integrated into the target site. The cell or organism is then grown to recover the organism with the targeted integration.

A selectable marker comprises a DNA segment that allows one to identify or select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like. Examples of selectable markers include, but are not limited to, DNA segments that comprise restriction enzyme sites; DNA segments that encode products which provide resistance against otherwise toxic compounds (e.g., antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT)); DNA segments that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA segments that encode products which can be readily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cell surface proteins); the generation of new primer sites for PCR (e.g., the juxtaposition of two DNA sequence not previously juxtaposed), the inclusion of DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; and, the inclusion of a DNA sequences required for a specific modification (e.g., methylation) that allows its identification.

Additional selectable markers include genes that confer resistance to herbicidal compounds, such as glyphosate, sulfonylureas, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillen and Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. The above list of selectable markers is not meant to be limiting. Any selectable marker can be used in the methods and compositions.

The activity of various promoters at a characterized location in the genome of a cell or an organism can be determined. Thus, the desired activity and/or expression level of a nucleotide sequence of interest can be achieved, as well as, the characterization of promoters for expression in the cell or the organism of interest.

In one embodiment, the method for assessing promoter activity in a cell or an organism comprises providing a cell or an organism comprising (e.g., in its genome) a target site having a first and a second recombination site, wherein the first and the second recombination sites are dissimilar and non-recombinogenic with respect to one another. A transfer cassette is introduced into the cell or the organism, where the transfer cassette comprises a promoter operably linked to a polynucleotide comprising a selectable marker and the transfer cassette is flanked by the first and the second recombination sites. At least one cell proliferation factor and a site-specific recombinase is provided, wherein the recombinase recognizes and implements recombination at the first and second recombination sites. Promoter activity is assessed by monitoring expression of the selectable marker. In this manner, different promoters can be integrated at the same position in the genome and their activity compared.

In some embodiments of the method for assessing promoter activity, the transfer cassette comprises in the following order: the first recombination site, a promoter operably linked to a third recombination site operably linked to a polynucleotide comprising a selectable marker, and the second recombination site, where the first, the second, and the third recombination sites are dissimilar and non-recombinogenic with respect to one another. This transfer cassette can be generically represented as RSa-P1::RSc::S1-RSb. Following the introduction of the transfer cassette at the target site, the activity of the promoter (P1) can be analyzed using methods known in the art. Once the activity of the promoter is characterized, additional transfer cassettes comprising a polynucleotide of interest flanked by the second and the third recombination site can be introduced into the organism. Upon recombination, the expression of the polynucleotide of interest will be regulated by the characterized promoter. Accordingly, organisms, such as plant lines, having promoters that achieve the desired expression levels in the desired tissues can be engineered so that nucleotide sequences of interest can be readily inserted downstream of the promoter and operably linked to the promoter and thereby expressed in a predictable manner.

It is further recognized that multiple promoters can be employed to regulate transcription at a single target site. In this method, the target site comprising the first and the second recombination sites is flanked by two convergent promoters. “Convergent promoters” refers to promoters that are oriented to face one another on either terminus of the target site. The same promoter, or different promoters may be used at the target site. Each of the convergent promoters is operably linked to either the first or the second recombination site. For example, the target site flanked by the convergent promoters can comprise P1→:R1-R2:←P2, where P is a promoter, the arrow indicates the direction of transcription, R is a recombination site, and the colon indicates the components are operably linked.

The transfer cassette employed with the target site having the convergent promoters can comprise, in the following order, the first recombination site, a first polynucleotide of interest orientated in the 5′ to 3′ direction, a second polynucleotide of interest orientated in the 3′ to 5′ direction, and a second recombination site. The insertion of the transfer cassette at the target site results in the first polynucleotide of interest operably linked to the first convergent promoter, and the second polynucleotide of interest operably linked to the second convergent promoter. The expression of the first and/or the second polynucleotide of interest may be increased or decreased in the cell or organism. The expression of the first and/or the second polynucleotide of interest may also be independently regulated depending upon which promoters are used. It is recognized that target sites can be flanked by other elements that influence transcription. For example, insulator elements can flank the target site to minimize position effects. See, for example, U.S. Publication No. 2005/0144665, herein incorporated by reference.

In further embodiments, methods are provided to identify a cis transcriptional regulatory region in an organism. By “transcriptional regulatory region” is intended any cis acting element that modulates the level of an RNA. Such elements include, but are not limited to, a promoter, an element of a promoter, an enhancer, an intron, or a terminator region that is capable of modulating the level of RNA in a cell. Thus, the methods find use in generating enhancer or promoter traps. In one embodiment, the reporter or marker gene of the target site is expressed only when it inserts close to (enhancer trap) or within (promoter trap) another gene. The expression pattern of the reporter gene will depend on the enhancer elements of the gene near or in which the reporter gene inserts. In this embodiment, the target site introduced into the cell or the organism can comprise a marker gene operably linked to a recombination site. In specific embodiments, the marker gene is flanked by dissimilar and non-recombinogenic recombination sites. The marker gene is either not operably linked to a promoter (promoter trap) or the marker gene is operably linked to a promoter that lacks enhancer elements (enhancer trap). Following insertion of the target site into the genome of the cell or the organism, the expression pattern of the marker gene is determined for each transformant. When a transformant with a marker gene expression pattern of interest is found, the enhancer/promoter trap sequences can be used as a probe to clone the gene that has that expression pattern, or alternatively to identify the promoter or enhancer regulating the expression. In addition, once a target site is integrated and under transcriptional control of a transcriptional regulatory element, methods can further be employed to introduce a transfer cassette having a polynucleotide of interest into that target in the cell or the organism. A recombination event between the target site and the transfer cassette will allow the nucleotide sequence of interest to come under the transcriptional control of the promoter and/or enhancer element. See, for example, Geisler et al. (2002) Plant Physiol 130:1747-1753; Topping et al. (1997) Plant Cell 10:1713-245; Friedrich et al. (1991) Genes Dev 5:1513-23; Dunn et al. (2003) Appl Environ Microbiol 1197-1205; and von Melchner et al. (1992) Genes Dev 6:919-27; all of which are herein incorporated by reference. In these methods, a cell proliferation factor (e.g., a babyboom polypeptide) is further introduced into the cell or organism to enhance recombination.

Further, methods are provided for locating preferred integration sites within the genome of a plant cell. Such methods comprise introducing into the plant cell a transfer cassette comprising in the following order: a first recombination site, a promoter active in the plant cell operably linked to a polynucleotide, and a second recombination site; wherein the first and second recombination sites are non-recombinogenic with respect to one another. A cell proliferation factor and site-specific recombinase that recognizes and implements recombination at the first and second recombination sites are introduced into the plant cell. The level of expression of the polynucleotide is determined using any method known in the art and the plant cell that is expressing the polynucleotide is selected.

Methods are also provided for the integration of multiple transfer cassettes at a target site in a cell. In some embodiments, the target site is constructed to have multiple sets of dissimilar and non-recombinogenic recombination sites. Thus, multiple genes or polynucleotides can be stacked or ordered. In specific embodiments, this method allows for the stacking of sequences of interest at precise locations in the genome of a cell or an organism. Likewise, once a target site has been established within a cell or an organism (for example, the target site can be stably integrated into the genome of the cell or organism), additional recombination sites may be introduced by incorporating such sites within the transfer cassette. Thus, once a target site has been established, it is possible to subsequently add sites or alter sites through recombination. Such methods are described in detail in International Application Publication No. WO 99/25821, herein incorporated by reference.

In one embodiment, the method comprises introducing into a cell having a target site comprising a first and a second recombination site a first transfer cassette comprising at least the first, a third, and the second recombination sites, wherein the first and the third recombination sites of the first transfer cassette flank a first polynucleotide of interest, and wherein the first, the second, and the third recombination sites are non-recombinogenic with respect to one another. Along with the first transfer cassette, a first site-specific recombinase is introduced into the cell, wherein the first site-specific recombinase recognizes and implements recombination at the first and the second recombination sites. A second transfer cassette is then introduced into the cell, comprising at least the second and the third recombination sites, wherein the second and the third recombination sites of the second transfer cassette flank a second polynucleotide of interest. In some embodiments, a single recombinase can recognize and implement recombination at the first and second recombination sites and at the second and third recombination sites. In other embodiments, along with the second transfer cassette, a second site-specific recombinase is introduced into the cell that recognizes and implements recombination at the second and the third recombination sites. The method further comprises introducing at least one cell proliferation factor to the cell before or during the introduction of the first recombinase, the second recombinase, or both the first and the second recombinase. In a related, alternative method, the target site of the cell has a target site comprising the first, second, and third recombination sites, the first transfer cassette comprises a first polynucleotide of interest flanked by the first and the second recombination sites, and the second transfer cassette comprises a second polynucleotide of interest flanked by at least the second and third recombination sites. A first and a second site-specific recombinase and a cell proliferation factor is introduced similar to the first method for the integration of multiple transfer cassettes described immediately above.

In other embodiments, methods are provided to minimize or eliminate expression resulting from random integration of DNA sequences into the genome of a cell or an organism, such as a plant. This method comprises providing a cell or an organism having stably incorporated into its genome a polynucleotide comprising the following components in the following order: a promoter active in the cell or the organism operably linked to an ATG translational start sequence operably linked to a target site comprising a first and a second functional recombination site, wherein the first and the second recombination sites are dissimilar and non-recombinogenic with respect to one another. A transfer cassette comprising a polynucleotide of interest flanked by the first and the second recombination site is introduced into the cell or the organism. The translational start sequence of the nucleotide sequence of interest in the transfer cassette has been replaced with the first recombination site. A cell proliferation factor (e.g., a babyboom polypeptide) and a recombinase is provided that recognizes and implements recombination at the recombination sites. Recombination with the target site results in the polynucleotide of interest being operably linked to the ATG translational start site of the target site contained in the polynucleotide. By operably linked is intended a fusion between adjacent elements and when used to refer to the linkage between a translational start a promoter and/or a recombination site implies that the sequences are put together to generate an inframe fusion that results in a properly expressed and functional gene product.

Methods for excising or inverting a polynucleotide of interest are provided. Such methods can comprise introducing into a cell having a target site comprising: a polynucleotide of interest flanked by a first and a second recombination site, wherein the first and the second sites are recombinogenic with respect to one another; at least one cell proliferation factor; and a double-strand break-inducing enzyme comprising a site-specific recombinase that recognizes and implements recombination at the first and the second recombination sites, thereby excising or inverting the polynucleotide of interest. Depending on the orientation of the recombination sites, the polynucleotide of interest will be excised or inverted when the appropriate recombinase is provided. For example, directly repeated recombination sites will allow for excision of the polynucleotide of interest and inverted repeats will allow for an inversion of the polynucleotide of interest.

The cell proliferation factor, double-strand break-inducing enzyme or a polynucleotide encoding the same, and in some embodiments, a transfer cassette, is introduced into a cell or an organism according to the presently disclosed methods. “Introducing” is intended to mean presenting to the organism, such as a plant, or the cell the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the organism or to the cell itself. The methods and compositions do not depend on a particular method for introducing a sequence into an organism, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the organism. Methods for introducing polynucleotides or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, virus-mediated methods, and sexual breeding.

“Stable transformation” means that the nucleotide construct introduced into a host cell or an organism integrates into the genome of the host and is capable of being inherited by the progeny thereof. “Transient transformation” is intended to mean that a polynucleotide is introduced and does not integrate into the genome of the host or that a polypeptide is introduced into a host.

Protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell being targeted. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. No. 4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and, U.S. Pat. No. 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

In specific embodiments, the sequences can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the double-strand break-inducing enzyme or cell proliferation protein or variants and fragments thereof directly into the plant or the introduction of a double-strand break-inducing enzyme or cell proliferation factor transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol. Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).

In other embodiments, the polynucleotide may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct within a viral DNA or RNA molecule. It is recognized that the double-strand break-inducing enzyme or cell proliferation factor may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.

The polynucleotides can be provided in a DNA construct. In addition, in specific embodiments, recognition sequences and/or the polynucleotide encoding an appropriate double-strand break-inducing enzyme is also contained in the DNA construct. The construct can include 5′ and 3′ regulatory sequences operably linked to the polynucleotide of interest. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. However, it is recognized that intervening sequences can be present between operably linked elements and not disrupt the functional linkage. For example, an operable linkage between a promoter and a polynucleotide of interest comprises a linkage that allows for the promoter sequence to initiate and mediate transcription of the polynucleotide of interest. When used to refer to the linkage between a translational start and a recombination site, the term operably linked implies that the sequences are put together to generate an inframe fusion that results in a properly expressed and functional gene product. Similarly, when used to refer to the linkage between a promoter and a recombination site, the linkage will allow for the promoter to transcribe a downstream nucleotide sequence. The cassette may additionally contain at least one additional gene to be introduced into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct may be provided with a plurality of restriction sites, recognition sequences, or recombination sites for insertion of the polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

In some embodiments, the DNA construct can include in the 5′ to 3′ direction of transcription, a transcriptional and translational initiation region, a polynucleotide of interest, and a transcriptional and translational termination region functional in the organism of interest.

The transcriptional initiation region, the promoter, may be native, analogous, foreign, or heterologous to the host organism, and/or to the polynucleotide of interest. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Such constructs may change expression levels of the polynucleotide of interest in the organism.

The termination region may be native or heterologous with the transcriptional initiation region, it may be native or heterologous with the operably linked polynucleotide of interest, or it may be native or heterologous with the host organism. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639. The polynucleotide of interest can also be native or analogous or foreign or heterologous to the host organism.

Sequence modifications in addition to codon optimization are known to enhance gene expression in a cellular host. These include elimination of spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

The DNA construct may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods or sequences known to enhance translation can also be utilized, for example, introns, and the like.

In preparing the DNA construct, the various DNA fragments may be manipulated, so as to place the sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

Generally, the DNA construct will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues and have been discussed in detail elsewhere herein.

A number of promoters can be used. As used herein “promoter” includes reference to a region of DNA involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in a plant cell. Any promoter can be used, and is typically selected based on the desired outcome (for a review of plant promoters, see Potenza et al. (2004) In Vitro Cell Dev Biol 40:1-22).

Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), the Agrobacterium nopaline synthase (NOS) promoter (Bevan et al. (1983) Nucl. Acids Res. 11:369-385), and the like. Other constitutive promoters are described in, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

In some embodiments, an inducible promoter can be used, such as from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also WO 99/43819, herein incorporated by reference. Promoters that are expressed locally at or near the site of pathogen infection include, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Mol Plant-Microbe Interact 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386 (nematode-inducible); and the references cited therein. Additional promoters include the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200). Wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nat Biotechnol 14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEES Lett 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6:141-150); and the like, herein incorporated by reference. Another inducible promoter is the maize In2-2 promoter (deVeylder et al. (2007) Plant Cell Physiol 38:568-577, herein incorporated by reference).

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. The promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners (De Veylder et al. (1997) Plant Cell Physiol. 38:568-77), the maize GST promoter (GST-II-27, WO 93/01294), which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, the PR-1 promoter (Cao et al. (2006) Plant Cell Reports 6:554-60), which is activated by BTH or benxo(1,2,3)thiaidazole-7-carbothioic acid s-methyl ester, the tobacco PR-1a promoter (Ono et al. (2004) Biosci. Biotechnol. Biochem. 68:803-7), which is activated by salicylic acid, the copper inducible ACE1 promoter (Mett et al. (1993) PNAS 90:4567-4571), the ethanol-inducible promoter AlcA (Caddick et al. (1988) Nature Biotechnol 16:177-80), an estradiol-inducible promoter (Bruce et al. (2000) Plant Cell 12:65-79), the XVE estradiol-inducible promoter (Zao et al. (2000) Plant J 24:265-273), the VGE methoxyfenozide inducible promoter (Padidam et al. (2003) Transgenic Res 12:101-109), and the TGV dexamethasone-inducible promoter (Bohner et al. (1999) Plant J 19:87-95). Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237; Gatz et al. (1992) Plant J 2:397-404; and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced expression of a sequence of interest within a particular plant tissue. Tissue-preferred promoters include Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Lam (1994) Results Probl. Cell Differ. 20:181-196; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.

Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12:255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35:773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23:1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90:9586-9590. In addition, promoter of cab and rubisco can also be used. See, for example, Simpson et al. (1958) EMBO J. 4:2723-2729 and Timko et al. (1988) Nature 318:57-58.

Root-preferred promoters are known and can be selected from the many available. See, for example, Hire et al. (1992) Plant Mol. Biol. 20:207-218 (soybean root-specific glutamine synthase gene); Keller and Baumgartner (1991) Plant Cell 3:1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14:433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3:11-22 (full-length cDNA clone encoding cytosolic glutamine synthase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2:633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Sci (Limerick) 79:69-76). Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2′ gene is root specific in the intact plant and stimulated by wounding in leaf tissue (see EMBO J. 8:343-350). The TR1′ gene, fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29:759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25:681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179. Another root-preferred promoter includes the promoter of the phaseolin gene (Murai et al. (1983) Science 23:476-482 and Sengopta-Gopalen et al. (1988) Proc. Natl. Acad. Sci. USA 82:3320-3324.

Seed-preferred promoters include both those promoters active during seed development as well as promoters active during seed germination. See Thompson et al. (1989) BioEssays 10:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and mi1ps (myo-inositol-1-phosphate synthase); (see WO 00/11177 and U.S. Pat. No. 6,225,529; herein incorporated by reference). For dicots, seed-preferred promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-preferred promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, nuc1, etc. See also WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed; herein incorporated by reference. In particular embodiments, the maize oleosin promoter set forth in SEQ ID NO: 55 or a variant or fragment thereof is used.

Where low-level expression is desired, weak promoters will be used. Generally, by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By low level is intended at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Alternatively, it is recognized that weak promoters also encompasses promoters that are expressed in only a few cells and not in others to give a total low level of expression. Where a promoter is expressed at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels. Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, and the like.

Other promoters of interest include the Rab16 promoter (Mundy et al. (1990) PNAS 87: 1406-1410), the Brassica LEA3-1 promoter (U.S. Application Publication No. US 2008/0244793), the HVA1s, Dhn8s, and Dhn4s from barley and the wsi18j, rab16Bj from rice (Xiao and Xue (2001) Plant Cell Rep 20:667-73), and D113 from cotton (Luo et al. (2008) Plant Cell Rep 27:707-717).

In some embodiments, the polynucleotide encoding a cell proliferation factor (e.g., babyboom polypeptide) is operably linked to a maize ubiquitin promoter or a maize oleosin promoter (e.g., SEQ ID NO: 65 or a variant or fragment thereof).

In some embodiments, the methods further comprise identifying cells comprising the modified target locus and recovering plants comprising the modified target locus. In some examples, recovering a plant having the modified target locus occurs at a higher frequency as compared to a control method without a cell proliferation factor.

Any method can be used to identify a plant cell or plant comprising a modified target locus. In some examples, plant cell or plants having a modified target locus are identified using one or more of the following techniques, including but not limited to PCR methods, hybridization methods such as Southern or Northern blots, restriction digest analyses, or DNA sequencing.

The cells having the introduced sequence may be grown into plants in accordance with conventional methods, see, for example, McCormick et al. (1986) Plant Cell Rep 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or with a different strain, and the resulting progeny expressing the desired phenotypic characteristic and/or comprising the introduced polynucleotide or polypeptide identified. Two or more generations may be grown to ensure that the polynucleotide is stably maintained and inherited, and seeds harvested. In this manner, transformed seed, also referred to as transgenic seed, having a polynucleotide, for example, comprising a modified target site, stably incorporated into their genome are provided.

In some embodiments, the activity and/or level of the cell proliferation factor (e.g., a babyboom polypeptide, Wuschel) is reduced prior to regenerating a plant from the plant cell having the modified target site. In some of these embodiments, the polynucleotide encoding the cell proliferation factor, and in particular embodiments, the polynucleotide encoding the double-strand break-inducing enzyme, as well, are excised prior to the regeneration of a plant. In some of these embodiments, the promoter and other regulatory elements that are operably linked to each of the heterologous polynucleotides are excised along with the heterologous polynucleotides. In certain embodiments, the polynucleotide encoding the cell proliferation factor (and in particular embodiments, the double-strand break-inducing enzyme) are flanked by recombination sites and an appropriate site-specific recombinase is introduced into the plant cell to excise the polynucleotide encoding the cell proliferation factor, and in some embodiments, the double-strand break-inducing enzyme, prior to regeneration of the plant cell into a plant. In some of those embodiments wherein both a babyboom polypeptide and a Wuschel polypeptide are provided to the plant cell, both the polynucleotide encoding the babyboom polypeptide and the polynucleotide encoding the Wuschel polypeptide are excised. The two polynucleotides can be present on the same or on different expression cassettes and, therefore, can be excised in one or two different excision reactions. In some of these embodiments, the polynucleotide encoding the site-specific recombinase for excising the babyboom and Wuschel polynucleotides can be located on the same expression cassette as the babyboom and Wuschel polynucleotides and all three polynucleotides can be excised through the activity of the site-specific recombinase.

In order to control the excision of the cell proliferation factor(s) (and in some embodiments, the double-strand break-inducing enzyme), the expression of the site-specific recombinase that is responsible for the excision can be controlled by a late embryo promoter or an inducible promoter. In some embodiments, the late embryo promoter is GZ (Uead et al. (1994) Mol Cell Biol 14:4350-4359), gamma-kafarin promoter (Mishra et al. (2008) Mol Biol Rep 35:81-88), Glb1 promoter (Liu et al. (1998) Plant Cell Reports 17:650-655), ZM-LEG1 (U.S. Pat. No. 7,211,712), EEP1 (U.S. Patent Application No. US 2007/0169226), B22E (Klemsdal et al. (1991) Mol Gen Genet 228:9-16), or EAP1 (U.S. Pat. No. 7,321,031). In some embodiments, the inducible promoter that regulates the expression of the site-specific recombinase is a heat-shock, light-induced promoter, a drought-inducible promoter, including but not limited to Hva1 (Straub et al. (1994) Plant Mol Biol 26:617-630), Dhn, and WSI18 (Xiao & Xue (2001) Plant Cell Rep 20:667-673). In other embodiments, expression of the site-specific recombinase is regulated by the maize rab17 promoter (nucleotides 1-558 or 51-558 of GenBank Acc. No. X1554 or active fragments or variants thereof; Vilardell et al. (1990) Plant Mol Biol 14:423-432; Vilardell et al. (1991) Plant Mol Biol 17:985-993; and U.S. Pat. Nos. 7,253,000 and 7,491,813; each of which is herein incorporated in its entirety), or a variant rab17 promoter (for example, the variant rab17 promoter set forth in SEQ ID NO: 54; see U.S. Provisional Application No. 61/291,257 and U.S. Utility Application entitled “Methods and compositions for the introduction and regulated expression of genes in plants,” filed concurrently herewith and herein incorporated by reference in its entirety). The wild type or modified rab17 promoter can be induced through exposure of the plant cell, callus, or plant to abscisic acid, sucrose, or desiccation. In some embodiments, the site-specific recombinase that excises the polynucleotide encoding the cell proliferation factor is FLP.

Also provided are compositions comprising plant cells or plants comprising a heterologous polynucleotide encoding a cell proliferation factor, wherein the plant cell or plant comprises a target site comprising a recognition sequence; a double-strand break-inducing enzyme that recognizes the recognition sequence; and a transfer cassette comprising a polynucleotide of interest and at least one region of homology with the target site. In some embodiments, the region of homology is a recognition sequence. In these embodiments, the double-strand break-inducing enzyme is a site-specific recombinase capable of recognizing and implementing recombination at the recombination sites within the target site and the transfer cassette. In certain embodiments, the target site is stably integrated into the plant genome.

In some embodiments, the cell proliferation factor is a member of the AP2 family of polypeptides. In some of these embodiments, the cell proliferation factor is a babyboom polypeptide, and in particular embodiments, the babyboom polypeptide comprises two AP2 domains and at least one of: SEQ ID NO: 9 or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 9; or SEQ ID NO: 12 or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 12. In particular embodiments, the cell proliferation factor has the sequence set forth in SEQ ID NO: 2, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 105, or 41 or has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 2, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 105, or 41. In some of these embodiments, both a babyboom polypeptide and a Wuschel polypeptide are provided to the plant cell.

In certain embodiments, the cell proliferation factor (e.g., babyboom polypeptide, Wuschel polypeptide) and/or the double-strand break-inducing enzyme is provided to the cell through the introduction of a polynucleotide encoding the cell proliferation factor and/or the double-strand break-inducing enzyme. In some of these embodiments, the polynucleotide encoding the cell proliferation factor has the sequence set forth in SEQ ID NO: 1, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 59, 101, 102, 103, 104, or 60 or has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 1, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 59, 101, 102, 103, 104, or 60. In some of these embodiments, the polynucleotide encoding the cell proliferation factor is operably linked to an oleosin or ubiquitin promoter. In some of those embodiments wherein a Wuschel polynucleotide is also introduced into the plant cell, expression of Wuschel is regulated by the NOS or In2-2 promoter.

The double-strand break-inducing enzyme can be an endonuclease, a zinc finger nuclease, a transposase, a topoisomerase, or a site-specific recombinase. In some embodiments, the double-strand break-inducing enzyme is an endonuclease or a modified endonuclease, such as a meganuclease. In other embodiments, the double-strand break-inducing enzyme is a site-specific recombinase such as FLP or Cre and the recognition sequence comprises a recombination site (e.g., FRT1, FRT87, lox). In some of these embodiments, the site-specific recombinase has the sequence set forth in SEQ ID NO: 43 (FLP) or 45 (Cre) or has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 43 or 45. In some of those embodiments wherein the site-specific recombinase is provided to the cell through the introduction of a polynucleotide that encodes the site-specific recombinase, the polynucleotide has the sequence set forth in SEQ ID NO: 42 (FLPm) or 44 (moCre) or has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 42 or 44.

In particular embodiments, the plant cell or plant comprises a heterologous polynucleotide of interest encoding a cell proliferation factor, wherein the plant cell or plant comprises a target site comprising a first recombination site, a nucleotide sequence, and a second recombination site; a transfer cassette comprising a third recombination site, a polynucleotide of interest, and a fourth recombination site, wherein the first and the third recombination sites are recombinogenic with respect to one another, and the second and fourth recombination sites are recombinogenic with respect to one another; and a site-specific recombinase capable of recognizing and implementing recombination at the first and third and second and fourth recombination sites.

The plant cell or plant can comprise more than one cell proliferation factor. For example, along with a babyboom polypeptide, the plant or plant cell can comprise a Wuschel polypeptide.

In particular embodiments, the heterologous polynucleotide encoding the cell proliferation factor comprises flanking recombination sites to facilitate its excision. In these embodiments, the plant further comprises a site-specific recombinase that recognizes the recombination sites flanking the heterologous polynucleotide encoding the cell proliferation factor. In some embodiments, this site-specific recombinase comprises FLPm or an active variant or fragment thereof. In some of those embodiments wherein the plant cell or plant further comprise a Wuschel polypeptide, the polynucleotide encoding the Wuschel polypeptide and the heterologous polynucleotide encoding the cell proliferation factor are flanked by recombination sites to facilitate the excision of both polynucleotides.

Any plant species can be transformed, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Peryea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), Arabidopsis, switchgrass, vegetables, ornamentals, grasses, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean and sugarcane plants are optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

As used herein, the term plant also includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.

In some of those embodiments wherein the organism to which the cell proliferation factor, double-strand break-inducing enzyme, and in certain embodiments, a transfer cassette, is a plant, these elements can be introduced into a plant cell. In particular embodiments, the plant cell is a cell of a recalcitrant tissue or plant, such as an elite maize inbred. As used herein, a “recalcitrant tissue” or “recalcitrant plant” is a tissue or a plant that has a low rate of transformation using traditional methods of transformation, such as those disclosed elsewhere herein. In some embodiments, the recalcitrant tissue or plant is unable to be transformed in the absence of the cell proliferation factor. In other embodiments, the recalcitrant tissue or plant has a rate of successful transformation of less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, less than about 0.1%, less than about 0.01%, less than about 0.001%, or less. Non-limiting examples of recalcitrant tissues include mature seed or mature seed tissue, a leaf or leaf tissue, a stem or stem tissue.

In some embodiments, the cell proliferation factor, double-strand break-inducing enzyme, and in certain embodiments, a transfer cassette, are introduced into a mature seed, mature seed tissue, or leaf tissue using the methods described in U.S. Provisional Application entitled “Methods and compositions for the introduction and regulated expression of genes in plants,” filed concurrently herewith.

Some embodiments of the methods provide for the targeted insertion of a polynucleotide of interest. If the polynucleotide of interest is introduced into an organism, it may impart various changes in the organism, particularly plants, including, but not limited to, modification of the fatty acid composition in the plant, altering the amino acid content of the plant, altering pathogen resistance, and the like. These results can be achieved by providing expression of heterologous products, increased expression of endogenous products in plants, or suppressed expression of endogenous produces in plants.

General categories of polynucleotides of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, those involved in biosynthetic pathways, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include sequences encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, oil, starch, carbohydrate, phytate, protein, nutrient, metabolism, digestability, kernel size, sucrose loading, and commercial products.

Traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Protein modifications to alter amino acid levels are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389 and WO 98/20122, herein incorporated by reference.

Insect resistance genes may encode resistance to pests such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); and the like.

Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the S4 and/or Hra mutations in ALS), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), genes providing resistance to glyphosate, such as GAT (glyphosate N-acetyltransferase; U.S. Pat. No. 6,395,485), EPSPS (enolpyruvylshikimate-3-phosphate synthase; U.S. Pat. Nos. 6,867,293, 5,188,642, 5,627,061), or GOX (glyphosate oxidoreductase; U.S. Pat. No. 5,463,175), or other such genes known in the art. The nptII gene encodes resistance to the antibiotics kanamycin and geneticin.

Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.

Commercial traits can also be encoded on a gene or genes that could, for example increase starch for ethanol production, or provide expression of proteins.

Reduction of the activity of specific genes (also known as gene silencing, or gene suppression) is desirable for several aspects of genetic engineering in plants. Many techniques for gene silencing are well known to one of skill in the art, including but not limited to antisense technology (see, e.g., Sheehy et al. (1988) Proc. Natl. Acad. Sci. USA 85:8805-8809; and U.S. Pat. Nos. 5,107,065; 5,453,566; and 5,759,829); cosuppression (e.g., Taylor (1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech. 8(12):340-344; Flavell (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Finnegan et al. (1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen. Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell 2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141; Zamore et al. (2000) Cell 101:25-33; Javier (2003) Nature 425:257-263; and, Montgomery et al. (1998) Proc. Natl. Acad. Sci. USA 95:15502-15507), virus-induced gene silencing (Burton, et al. (2000) Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio. 2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature 334: 585-591); hairpin structures (Smith et al. (2000) Nature 407:319-320; WO 99/53050; WO 02/00904; and WO 98/53083); ribozymes (Steinecke et al. (1992) EMBO J. 11:1525; U.S. Pat. No. 4,987,071; and, Perriman et al. (1993) Antisense Res. Dev. 3:253); oligonucleotide mediated targeted modification (e.g., WO 03/076574 and WO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO 03/048345; and WO 00/42219); and other methods or combinations of the above methods known to those of skill in the art.

The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, and, (d) “percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et at (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as ³²P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the babyboom polynucleotide. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

For example, the entire babyboom polynucleotide, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding babyboom polynucleotide and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among babyboom polynucleotide sequences and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length. Such probes may be used to amplify corresponding babyboom polynucleotide from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T_(m) can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T_(m) is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m), hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the T_(m) can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T_(m)) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T_(m)); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T_(m)); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)). Using the equation, hybridization and wash compositions, and desired T_(m), those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T_(m) of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley—Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a polypeptide” is understood to represent one or more polypeptides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

Throughout this specification and the claims, the words “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise.

As used herein, the term “about,” when referring to a value is meant to encompass variations of, in some embodiments±50%, in some embodiments±20%, in some embodiments±10%, in some embodiments±5%, in some embodiments±1%, in some embodiments±0.5%, and in some embodiments±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the presently disclosed subject matter be limited to the specific values recited when defining a range.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL Example 1 Vector Construction

Maize recombination targets (RTL) were created using Agrobacterium transformation of immature maize embryos (Ishida et al. (1996) Nat Biotechnol 14:745-750). The LBA4404 Agrobacterium strain was used, which carried a specialized binary T-DNA plasmid system (Komari et al. (1996) Plant J 10:165-174) developed for high efficiency maize transformation. The binary Agrobacterium plasmid PHP21199 (similar to pSB124, Komari et al. (1996)), which is a T-DNA containing derivative of plasmid PHP10523 (similar to pSB1, Komari et al. (1996)) was constructed as follows. Visual and selectable marker genes were built into the T-DNA region of the intermediate construct, PHP21198 (similar to pSB12, Komari et al. (1996)), and then introduced into Agrobacterium to create the co-integrated binary plasmid, PHP21199. The selectable marker expression cassette in the PHP21199 plasmid consisted of the maize ubiquitin) (UBI) promoter (Christensen & Quail (1996) Transgenic Res 5:213-218), 5′ untranslated region (5′ utr), and intron (UBI PRO), a sequence encoding glyphosate n-acetyltransferase (GAT4602) (Siehl et al. (2007) J Biol Chem 282:11446-11455), and a 3′ region from the protease inhibitor 2 (PINII) gene of potato. The visual marker expression cassette in the PHP21199 plasmid consisted of the yellow fluorescent protein (YFP) gene (zs-yellow1 n1) (Clontech, Palo Alto, Calif.) expressed by the same promoter and terminator elements as the gat gene (UBI PRO, PINII). The wild-type FRT was inserted between the maize ubiquitin promoter and the YFP gene. The selectable and visual marker expression cassettes, as well as the properly positioned FRT sites, were assembled with the multi-site Gateway® (Invitrogen, Carlsbad, Calif.) system. The plasmid backbone of PHP21198 served as the destination plasmid (pDEST) with the destination site between the RB and LB in the T-DNA region and three Gateway® entry vectors (pDONR) were provided; one for each marker gene and one for the downstream FRT87 recombinase site. The FRT87 recombinase site is located 3′ of the final PINII 3′ region. The PHP21199 plasmid therefore comprised RB-UBI PRO::FRT1::YFP+UBI PRO::GAT4602::FRT87-LB.

Site-specific integration (SSI) donor plasmids PHP22297 and PHP27064 were built using the multi-site Gateway® (Invitrogen) system using methods similar to those used to construct the PHP21198 vector, except that an Agrobacterium vector was not used since the donor plasmids were introduced into plant cells by particle bombardment. Instead, the destination site was provided by the commercially available pDEST R4-R3 vector (Invitrogen). The entry vector for the first position of PHP22297 consisted of a promoterless bar gene with the PINII terminator. In place of the promoter is a copy of the 35S cauliflower mosaic virus (CaMV 35S) termination region. This feature was included for the purpose of reducing potential bar gene expression due to random promoter trapping following donor integration into the plant genome outside the target site. The FRT1 site was placed between the CaMV 35S terminator and the bar gene to match the FRT1 in the target constructs and integrations. The second entry vector contained a cyan fluorescent protein (CFP) visual marker (am-cyan 1) (Clontech) operably linked to maize UBI PRO and PINII 3′ regions as described above. The FRT87 site was placed in the third and final entry vector in order to position the site downstream of all the genes in the donor construct and to match the FRT87 position in the target construct. PHP22297 comprises FRT1::BAR+UBI PRO::CFP::FRT87. Donor construct PHP27064 was also constructed using pDEST R4-R3 (Invitrogen). The first entry vector was nearly identical to that for PHP22297 except that the bar gene was replaced by GAT4621, a GAT gene variant with similar but improved function to GAT4602. This entry vector did not include the 35S CaMV terminator region upstream of the promoterless gat gene. The second entry vector for PHP27064 had YFP in place of CFP, along with the same expression elements as the second entry vector used in the construction of PHP22297. The third entry vector included only FRT87 and was the same as that used for PHP22297. PHP27064 comprises FRT1::GAT4621+UBI PRO::YFP::FRT87.

Example 2 Recombinant Target Lines (RTL)

Zea mays immature embryos were transformed by a modified Agrobacterium-mediated transformation procedure (Djukanovic et al. (2006) Plant Biotechnol J 4:345-357) to introduce the T-DNA from PHP21199. Briefly, 10-12 days after pollination (DAP) embryos were dissected from sterile kernels and placed into liquid medium. After embryo collection, the medium was replaced with 1 ml of Agrobacterium suspension at a concentration of 0.35-0.45 OD at 550 nm, wherein the Agrobacterium comprised the T-DNA. After a five minute incubation at room temperature, the embryo suspension was poured onto a media plate. Embryos were incubated in the dark for 3 days at 20° C., followed by a 4 day incubation in the dark at 28° C. and a subsequent transfer onto new media plates containing 0.1778 mg/L glyphosate and 100 mg/L carbenicillin. Embryos were subcultured every three weeks until transgenic events were identified. Regeneration was induced by transferring small sectors of tissue onto maturation media containing 0.1 μM ABA, 0.5 ml/L zeatin, 0.1778 mg/L glyphosate, and 100 mg/L carbenicillin. The plates were incubated in the dark for two weeks at 28° C. Somatic embryos were transferred onto media containing 2.15 g/L MS salts (Gibco 11117: Gibco, Grand Island, N.Y.), 2.5 ml/L MS Vitamins Stock Solution, 50 mg/L myo-inositol, 15.0 g/L sucrose, 0.1778 mg/L glyphosate, and 3.0 g/L Gelrite, pH 5.6 and incubated under artificial light at 28° C. One week later, plantlets were moved into glass tubes containing the same medium and grown until they were sampled and/or transplanted to soil. Target lines were screened by qPCR to assess the copy number of the transgenes and only single copy integration events were used as targets.

Example 3 Transformation and Regeneration of Recombinase-mediated Cassette Exchange (RMCE) Events

Two plasmids were typically co-bombarded with SSI donor plasmids to facilitate recombination in PHWWE: PHP5096 and PHP21875. PHP5096 included a maize codon-optimized flp recombinase gene (SEQ ID NO: 42) under the control of maize UBI PRO and a pinII 3′ sequence. The second co-bombarded plasmid, PHP21875, contained a maize odp2 gene (also referred to herein as maize BBM; see WO 2005/075655, which is herein incorporated by reference in its entirety) controlled by the maize UBI PRO and pinII terminator. Three plasmids were typically co-bombarded with SSI donor plasmids to facilitate recombination in PHI581. The FLP plasmid was PHP5096 as above, but the second plasmid with BBM is either PHP21875 or PHP31729 with BBM expression regulated by the maize oleosin promoter (OLE). The third plasmid introduced into PHI581 is PHP21139, which has an auxin-inducible promoter IN2-2 controlling the expression of the maize wuschel gene (ZmWUS2). Experiments were performed with or without the BBM expression cassette to assess its impact on the recovery of RMCE events.

i) Delivery of Donor Vector

The donor plasmid was delivered via biolistic-mediated transformation into hemizygous immature embryos containing the recombinant target site created by the integration of PHP21199. 9 toll DAP immature embryos (1-1.5 mm in size) dissected from sterilized kernels were plated with their axis down onto media comprising 4.0 g/L N6 Basal salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/L thiamine HCl, 1.5 mg/L 2,4-D, 0.690 g/L L-proline, 30 g/L sucrose, 0.85 mg/L silver nitrate, and 3.0 g/L Gelrite, pH 5.8 and incubated in the dark at 28° C. for 3 to 5 days before the introduction of DNA. Two to four hours prior to bombardment, the embryos were plasmolyzed by placing them on the above media containing 120 gm/L of sucrose.

Plasmid DNA was associated with gold particles in preparation for biolistic-mediated transformation by mixing 100 μg of the donor plasmid, 10 μg of PHP5096 (encoding for mFLP), and in some bombardments, 10 μg of the helper plasmid PHP21875 (UBI:ODP2) (the volume of the DNA solution was adjusted to 40 μl), 50 μl of 1-μm gold particles at 0.01 mg/μl, and 5 μl TFX-50 (Promega E1811/2). The solution was allowed to gently mix for 10 minutes. The particles and attached DNA were spun down for 1 minute at 10,000 rpm and then the supernatant was removed and replaced with 120 μl of 100% ethanol. The particles were then re-suspended by gentle sonication. 10 μl of the particle solution was spotted on each carrier disc and the ethanol was allowed to evaporate. The macro carrier was placed 2.5 cm from a 450 psi rupture disc with the immature embryos placed on a shelf 7.5 cm below the launch assembly.

ii) Selection of RMCE Events

After bombardment, the embryos were removed from the high sucrose media and placed back on the same medium containing 30 g/L sucrose. The embryos were incubated in the dark at 28° C. for 7 days, at which time the embryos were moved to selection plates of the above media containing either 3.0 mg/L bialaphos (selection of first round RMCE events) or 0.1778 mg/L glyphosate (selection of second round RMCE events). Embryos were subcultured to fresh medium after 3 weeks and transgenic events were identified 4 weeks later. Transgenic events growing under selection were then observed for their fluorescent phenotype. Those that exhibited a fluorescent phenotype indicative of RMCE were regenerated under the appropriate selective agent (bialophos or glyphosate) using the above protocol. Plantlets were sampled and/or transplanted to soil.

iii) Regeneration

Plant regeneration medium (288J) comprised 4.3 g/L MS salts (GIBCO 11117-074), 5.0 ml/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/L thiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L glycine brought to volume with polished D-I H2O) (Murashige & Skoog (1962) Physiol Plant 15:473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/L sucrose and 1.0 ml/L of 0.1 mM abscisic acid (brought to volume with polished D-I H2O after adjusting to pH 5.6), 3.0 g/L Gelrite™ (added after bringing to volume with D-I H2O), and 1.0 mg/L indoleacetic acid and 3.0 mg/L bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprised 4.3 g/L MS salts (GIBCO 11117-074), 5.0 ml/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/L thiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L glycine (brought to volume with polished D-I H2O), 0.1 g/L myo-inositol, 40.0 g/L sucrose (brought to volume with polished D-I H2O after adjusting pH to 5.6); and 6 g/L bacto-agar (added after bringing to volume with polished D-I H2O), and was sterilized and cooled to 60° C.

iv) Polymerase Chain Reaction

DNA was extracted via a modified alkaline lysis method using 1 punch (200 ng) of fresh leaf tissue (Truett et al. (2000) Biotechniques 29:52-54). For quantitative PCR (qPCR), each gene was quantitated using specific forward and reverse primers along with a corresponding FAM based MGB (Applied Biosystems, Foster City, Calif.) fluorogenic multiplexed probe. Each assay was primer titrated and normalized to an amplification signal from an endogenous gene which utilized a VIC®-based sequence specific probe and primer set. The amplification reactions for the bar and CFP genes were run simultaneously with the normalizing gene in a single tube reaction. Upon completion of the qPCR, all raw data were used to calculate the dCT values. Copy number determination was computed with the ΔΔCT method as described in the ABI User Bulletin #2 (Applied Biosystems, Foster City, Calif.). Endpoint positive and negative qPCR calls were made for flp, ubi:odp2, ubi:frt1:bar and the FrtX junctions according to the dCT estimates. A PCR reaction requiring 5 additional cycles than the normalizing gene was considered negative for the transcript.

v) Sequencing

QPCR samples identified as positive for recombinant junctions (UBI-FRT1-BAR, donor-FRT87-target) were further characterized by agarose gel electrophoresis (FIG. 5) and sequencing. Each qPCR reaction was run as an individual lane on a 2% agarose gel and visualized by ethidium bromide staining under UV light. DNA bands of the expected size were independently cut from each lane of the gel and extracted from the agarose using the QiaQUICK gel extraction kit (Qiagen, Valencia, Calif.). Samples of these extractions were submitted directly for DNA sequencing. Replicate DNA samples were submitted for sequencing with both forward and reverse sequencing primers.

vi) Southern Blots

Leaf tissue (2-10 grams fresh weight) was freeze-dried and ground to a fine powder. Ground tissue (350 mg) was re-suspended in 9 ml CTAB extraction buffer with β-mercaptoethanol (10 μl/ml). This solution was incubated at 65° C. for 1 hour. Every 20 minutes, tubes were inverted several times to mix the material and solution. Tubes were removed from the incubator and allowed to cool 10 minutes prior to adding 5 ml chloroform/octanol (24:1). Tubes were mixed by gently inverting for 5 minutes, and then centrifuged at 2500-3000 rpm (1100×G) for 30 minutes. The aqueous top layer was transferred to a fresh tube containing 11 ml precipitation buffer, and inverted several times gently. The tubes were allowed to stand at 25° C. (room temperature) for 30 minutes to 2 hours, were centrifuged at 2000 rpm for 20 minutes, and the supernatant was discarded. The tubes were inverted to dry the pellet. The dried pellet was completely dissolved in 2 ml of 100 mM Tris (pH 7.5), 10 mM EDTA (pH 7.5), 0.7 M NaCl, and precipitated in 5 ml of 95-100% ethanol. DNA was pipetted into a tube containing 1 ml of 76% ethanol, 0.2 M sodium acetate for 20 minutes, transferred to a fresh tube containing 1 ml 76% EtOH, 10 mM ammonium acetate for 1 minute, and then transferred again into a third tube and re-suspended.

Example 4 Transient Expression of ZmBBM and Recovery of RMCE Events in Maize

Recombinant Target Loci (RTL) were created by Agrobacterium-mediated transformation of immature maize embryos. The target sequence was flanked on the 5′ side by the wild-type FLP recognition target site (FRT1) paired on the 3′ side with a heterospecific FRT87. The integration copy number was determined by real-time quantitative PCR (qPCR) and transgenic events containing only a single RTL with a single copy of each gene were used. The RTL contained a yellow fluorescent protein gene (YFP) driven by the maize ubiquitin promoter. The wild-type FRT was inserted between the maize ubiquitin promoter and the YFP gene to act as a promoter trap for activation of a promoterless marker gene in the donor vector following FLP-mediated recombination at the FRT site. The target vectors also contained the selectable marker gene glyphosate acetyltransferase (GAT) driven by the maize ubiquitin promoter.

Immature embryos containing the RTL were re-transformed by particle bombardment, wherein the donor vector was co-delivered with the vector PHP5096 (UBI PRO::FLPm::pinII) in all experiments along with the helper plasmid PHP21875 (UBI PRO::ZmBBM::pinII) in the majority of experiments, both at 1/10 of the concentration of the donor vector. In this instance, transient expression of FLP and BBM was achieved through a reduction in the titer of both the FLP and BBM-containing plasmids, while effectively eliminating random integration and subsequent stable expression of both cassettes. Other means of promoting transient expression can also be used, such as delivery of FLP and/or BBM RNA or protein, in addition to the standard amount of donor plasmid as the substrate for RMCE.

In the first round of RMCE, the donor sequence, flanked by FRT1 and FRT87 sites, contained a promoterless bar gene and the gene encoding the cyan fluorescent protein (CFP) controlled by the maize ubiquitin promoter. RMCE resulted in the exchange of the YFP and GAT genes located at the RTL with bar and CFP from the donor plasmid. To demonstrate the ability to reuse a target site with the FLP/FRT recombination system, a second round of RMCE was performed. Two RTLs were chosen that contained the FRT1-FRT87 pair. The product of the first round of RMCE at the RTL became the target for a new round of RMCE. The next round of RMCE was initiated by delivering the PHP27064 donor vector by particle bombardment. The donor vector contained the wild type FRT1, a promoterless GAT gene for selection and Ubi:YFP flanked by the heterospecific FRT87. RMCE resulted in the exchange of the bar and CFP genes located at the RTL with GAT and YFP from the donor plasmid. The FLP protein used to mediate the recombination was again transiently expressed by co-delivery of the vector PHP5096.

In the first round of RMCE, replacement of the target sequence at the RTL by the donor sequence led to expression of the otherwise promoterless bar gene. Putative RMCE events were initially selected by placing bombarded embryos on bialaphos-containing media (Table 1, column 2). Growth of callus on bialaphos-containing media was indicative of site-specific integration, but some random integrations of the donor vector also resulted in expression of the promoterless bar gene. In fact, random integration of the donor plasmid and growth on bialaphos-containing media was more frequent than RMCE. On average, under our experimental conditions, 9 bialaphos-resistant calli were routinely recovered for every 1 RMCE event identified. Nevertheless, use of the promoter trap and selection on bialaphos-containing media enriched the population of selected calli for RMCE events.

Calli growing on bialaphos-containing media were further characterized by phenotypic loss and gain of expression of fluorescence marker genes. In the first round of RMCE, the excision of the YFP gene resulted in calli which were negative for the YFP phenotype, while integration (targeted or random) of CFP contained in the donor vector, resulted in expression of CFP. In contrast, random integration of the donor vector did not result in replacement and calli were positive for YFP.

In the second round of RMCE, activation of a promoterless GAT gene (in the donor cassette) was used to chemically select for RMCE prior to monitoring of the fluorescent phenotype. In this case, putative RMCE events were YFP positive due to the integration of the donor cassette and CFP negative due to the exchange and excision of the FRT flanked sequence at the RTL. Callus sectors showing the expected fluorescence pattern were transferred to plant regeneration media.

Molecular confirmation of RMCE was performed on DNA extracted from regenerated plantlets. Putative RMCE events were characterized with a series of six PCR reactions. PCR primers unique to the target and donor sequences were used in combination to amplify DNA fragments bridging the recombined FRT junctions. PCR amplification was observed only when recombination between FRT sites at the RTL and donor occurred. Routinely, real-time quantitative PCR was used for this analysis. To verify that the PCR product was generated across the recombinant junction, a sample of the qPCR products were run out on a gel to demonstrate size and sequenced to demonstrate the presence of target sequence, the FRT site, and donor sequence. The predicted fragment sizes of the recombinant products were confirmed by Southern blot hybridization. Putative RMCE events were analyzed by real-time quantitative PCR for copy number of genes in the donor cassette. Excision of the target sequence was verified by qPCR for the fluorescent marker gene initially at the RTL. QPCR was also used to determine if the FLPm or ODP2 genes had integrated.

As can be seen in Table 1, RMCE events were identified through a sieving process, first by activation of a promoterless selectable marker, then by phenotyping of fluorescence and finally by molecular analysis of regenerated plants. Samples found to have both recombinant FRT junctions and excision of the target sequence were considered to be the result of RMCE.

As another means of confirming recombination, genomic DNA was extracted from several of the SSI events and sequenced across the FRT junctions to demonstrate the presence of both target and donor sequence and conservation of the FRT site itself. In one of the recombinant events, sequencing of the FRT87 site revealed a mutation in the 8 bp core region of the FRT site. The number of copies of integrated donor genes was determined by qPCR. Excision of the target sequence was verified by qPCR for the fluorescent marker gene initially at the RTL. qPCR was also used to determine if the FLPm gene had integrated. Random integrants growing under selection and not expressing the target fluorescent marker were identified and eliminated based on the lack of PCR products for the FRT junctions (Table 1, column 3). Precise RMCE was identified by the pattern of the PCR results (Table 1, columns 4 and 5). Only those events containing both the 5′ and 3′ FRT junctions, a single copy of the donor cassette and the absence of the target sequence and FLPm were considered precise RMCE events (Table 2). An RMCE event was considered imprecise if it contained more then a single copy of either of the donor genes even though both FRT junctions were present. Of the events found to have recombined at both FRT sites, about 10% also contained a random integration locus which segregated independently in the next generation. Various other types of imprecise RMCE and site-specific integrations were also identified by molecular characterization. In all, forty precise RMCE events were identified in the first round of RMCE.

TABLE 1 Identification of RMCE events in re-transformed embryos. Regenerable, Random Site-specific RMCE (Both Target Bialaphos bialaphos integration integration recombinant embryos resistant resistant, (No recombinant (Recombinant FRT1 and FRT bombarded calli CFP+/YFP− FRT junction) junction only) junctions) 14,945 560 129 56 21 52 3.75% * 0.86% 0.37% 0.14% 0.35% * Percent of bombarded embryos

Although events were identified in which FRT sites in the donor cassette recombined with those at the RTL, not all resulted in clean RMCE events (Table 2). Of the 52 events that had recombination of both FRT sites and loss of the target sequence (RMCE), 12 were found to have additional integrations of the donor cassette or integration of FLP or ZmBBM. Recombination was observed to occur at only the FRT1 site resulting in the separation of YFP from the ubiqutin promoter with and without the excision of the entire target sequence. Random integration of the donor cassette, as observed previously, would result in growth under selection with loss of YFP expression due to excision of the target sequence by illegitimate recombination between heterospecific FRT sites or silencing of YFP.

TABLE 2 Genotyping of putative RMCE plantlets by real time quantitative PCR. bar CFP FRT1 FRT87 (est. (est. # Integration junction junction copy) copy) YFP FLPm events Desired recombination product + + 1 1 − − 40 (Clean RMCE) Other patterns of integration observed RMCE - with additional donor + + ≧1 ≧1 − +/− 12 cassette and/or integrated FLP or ZmBBM plasmid FRT1 recombination only - target + − ≧1 ≧1 − +/− 16 sequence excised FRT1 recombination only - target + − ≧1 ≧1 + +/− 5 sequence not excised Random integration - target − − ≧1 ≧1 − +/− 12 sequence excised Random integration - target − − ≧1 ≧1 + +/− 31 sequence not excised Unknown - Complex integration +/− +/− ≧1 ≧1 +/− +/− 13

About 30% of the regenerated events selected by phenotype (bialaphos resistant, CFP positive, YFP negative) were precise RMCE events based on molecular characterization, while about 70% of the regenerated events were eliminated. In ˜60% of the discarded events, the FRT junctions were not found. These events may be the result of random integration of the donor plasmid. The remaining 40% of the discarded events appeared to have undergone site-specific integration at the target locus, but resulted in integration patterns reflecting either recombination at only the FRT1 site or an imprecise RMCE (Table 2). In a few events, FLPm was found to be integrated, but these events generally had other abnormalities.

In the second round of RMCE, activation of a promoterless GAT gene in the donor sequence was used to select for RMCE. In this case, about 62.5% of the regenerated events selected by phenotype were precise RMCE events based on molecular characterization. 96% of the putative RMCE events selected based on phenotype that reached the plant stage were found to have recombined at least at FRT1. The frequency of single recombination events at FRT1 and imprecise RMCE was 45% in the first round of RMCE and 38% in the second round.

The PCR reactions crossing the FRT junctions that were used to identify RMCE events were verified by both sequencing the PCR products and by Southern blot hybridization. The PCR products derived from several events were sequenced to demonstrate the contribution of sequence from the target and donor flanking the FRT site. RMCE was also verified by Southern blot hybridization of genomic DNA extracted from 30 putative RMCE events.

In the above experiments, an equal number of non-ZmBBM and ZmBBM treatments were not analyzed, but embryos from many ears were evaluated from both treatments. Overall, inclusion of ZmBBM resulted in a general 2-3 fold improvement in RMCE recovery in maize as compared to experiments in which the ZmBBM expression cassette was not used.

Example 5 Controlled Expression of ZmBBM

Any method can be used to control the timing and or location of expression of a cell proliferation factor, for example, ZmBBM. Molecular cloning and vector construction methods are well known and any such methods can be used to generate constructs with various elements or systems to regulate the timing or location of expression.

A. Transient Expression of ZmBBM

A particle gun was used to deliver the donor plasmid PHP22297 and PHP5096 plus or minus a UBI PRO::ZmBBM::pinII containing plasmid (PHP21875). During the TFX-mediated precipitation, 100 ng of PHP22297 and 10 ng of PHP5096 and PHP21875 (in the ZmBBM-containing treatment) were mixed. These plasmids, attached to gold particles as described in Example 3, were shot into immature embryos containing a single integrated copy of the T-DNA from PHP21199 (the target locus for RMCE). For this comparison (plus or minus ZmBBM), equal numbers of embryos from each ear, for a total of 176 ears, were used for side-by-side testing. For the control treatment (minus ZmBBM), 4551 bombarded embryos were taken through the selection protocol, and 13 RMCE events were recovered for an overall frequency of 0.29%. When ZmBBM was included in the bombardment, 4719 embryos produced 29 RMCE events for an overall frequency of 0.61%. This represented a consistent 2-fold increase in RCME recovery when the ZmBBM gene was included.

B. Tissue-Preferred Expression of ZmBBM

The ZmBBM gene was placed under the control of a maize oleosin promoter (SEQ ID NO: 55), which is a seed-preferred promoter expressed only in the scutella of developing embryos. The resulting expression plasmid containing OLE PRO::ZmBBM::pinII (PHP31729) was co-delivered along with the donor vector PHP22297, into immature embryos containing a single copy of the recombination target locus. Following selection on bialaphos and screening for loss of YFP and gain of CFP, RMCE events have been recovered. Expression of ZmBBM in callus cells increases the frequency of RMCE.

C. Excision of ZmBBM

An excisable ZmBBM plasmid comprising two expression cassettes (loxP-Ubi::ZmBBM::pinII+Rab17::Cre-loxP) is created. These two expression cassettes are co-delivered, along with the donor vector PHP22297, into immature embryos containing a single copy of the Recombination Target Locus. Expression of ZmBBM in callus cells increases the frequency of RMCE. In these experiments, the promoter controlling the expression of Cre is inactive during callus growth and chemical selection of RMCE events. Upon mild desiccation of the callus, for example, by placing the callus on high osmoticum such as 18% sucrose or onto dry filter papers for 1-3 days, expression of Cre recombinase is stimulated and both the BBM and Cre expression cassettes, being flanked by loxP recombinase target sites, are excised. Regeneration of fertile RMCE events is performed as described elsewhere herein.

D. Inducible Expression of ZmBBM for Recovery of RMCE Events in Maize

The ZmBBM gene can be placed under the control of an inducible expression system, such as that described in U.S. Application Publication No. 2008/0201806 A1, which is herein incorporated by reference in its entirety. Expression cassettes comprising a Triple-Op 35S promoter (Gatz et al. (1992) Plant J 2:397-404) and a pinII 3′ sequence operably linked to the ZmBBM gene and a UBI PRO-driven maize-codon modified Tet repressor are constructed. These expression cassettes are co-delivered, along with the donor vector PHP22297, into immature embryos containing a single copy of the Recombination Target Locus. The addition of 1 mg/L tetracycline to the culture medium resulting in BBM expression stimulates cell division and results in an increased recovery of RMCE events in maize.

E. Co-Expression of BBM and Wuschel

Developmental and inducible promoters were combined to control the expression of ZmBBM and ZmWUS2, respectively, in order to accomplish site specific integration (SSI) in maize inbred PH581. The experiments involved a different SSI target plasmid, PHP17797, although the basic function was identical to PHP21199 as described above. PHP17797 has the maize ubiquitin promoter driving FLP recombinase as the first gene that included the wild type FRT (FRT1) recombinase site. The second gene was CAMV35S PRO:BAR: pinII to provide bialaphos resistance in tissue culture. After the BAR gene, the FRT5 recombinase site was used instead of the FRT87 in PHP21199. Target immature embryos (PH581, 13 DAP) were bombarded using the particle gun for the co-delivery of donor constructs and developmental gene constructs. The ultimate goal was to recover normal fertile plants and then to segregate BBM and WUS2 from the transformation construct in the progeny. SSI donor vector, PHP33552, was bombarded with and without developmental gene constructs to compare the effect of including BBM and WUS2. PHP33552 included a promoterless gene encoding the yellow fluorescent protein (YFP, ZS-Yellow1 N1, Clontech, Palo Alto, Calif., USA). The genes in PHP33552 were flanked by FRT1 and FRT5 to facilitate recombinase-mediated cassette exchange (RMCE) in the presence of FLP recombinase. Correct site-specific integration activates YFP from a captured promoter in the target locus.

Using a particle gun for transformation, both SSI and standard transformation was attempted in SSI target lines without added BBM and/or WUS2 constructs. PH581 was capable of developing a low frequency of callus using standard transformation methods (0.3%) and a few events were regenerated. The regenerated plants were recovered to the greenhouse and set seed. When SSI methods were used, the numbers of transformed calli with the correct phenotype were lower than with standard transformation methods and no plants could be regenerated. PH581 plant regeneration from tissue culture occurs at a relatively low frequency compared to model maize lines for transformation, such as the public line Hi-II.

Constitutively expressed BBM and WUS2 were co-bombarded with donor vectors for SSI. In these experiments, the maize Ubi promoter controlled the expression of ZmBBM and the Agrobacterium nopaline synthase (NOS) promoter regulated ZmWUS2 expression. These treatments provided a higher frequency of callus with the SSI phenotype (10-30%). SSI was confirmed by real-time quantitative PCR (QPCR) analysis in callus that demonstrated continued growth in culture and exhibited the expected phenotype. Importantly, plants were able to be regenerated from the SSI positive callus. However, the plants demonstrated abnormal morphology, suspected to be due at least in part to the uncontrolled expression of BBM and WUS2. Roots showed the thickened phenotype attributable to BBM expression. As in past experiments with these developmental genes, regeneration frequency is negatively impacted by BBM and WUS2 expressed in this manner.

In another set of particle gun transformation experiments using immature PH581 embryos from SSI target lines, standard transformation and SSI were tested with the controlled expression of ZmBBM and ZmWUS2. The maize embryo-preferred promoter, oleosin (Ole Pro) was employed to regulate ZmBBM expression. This promoter is active in developing embryos during callus growth and kernel development. The maize IN2-2 PRO (deVeylder et al. (2007) Plant Cell Physiol 38:568-77) was used to express ZmWUS2. The IN2-2 PRO promoter has a low level constitutive activity, which can be further activated in the presence of auxin that can be provided in the tissue culture medium. This expression strategy allowed for the recovery of a number of callus events having the SSI phenotype. It also provided for the recovery of young T0 plants that were characterized with multiple qPCR assays to demonstrate SSI and to confirm the presence or absence of target genes, extra copies of genes from PHP33552, and integrated copies of the BBM and WUS2 plasmids. Young plants with the correct qPCR profile and YFP phenotype were advanced to the greenhouse where they developed into late-stage plants. In most cases, these plants were fertile. In some instances, plants exhibited delayed development or a stunted phenotype. During the flowering stage, the segregation of the cell proliferation transgenes was promoted by crossing tissue cultured plants with conventional PH581. Ears were harvested at about 13-15 DAP and immature embryos were plated on basic culture medium for embryo rescue. YFP positive kernels segregated 1:1 with null kernels as predicted when accounting for single, unlinked transgenic loci, one of which carries OLE PRO-ZmBBM and the second a recombined target locus. QPCR analysis of progeny plants confirmed that the YFP positive plants contained a recombinant SSI target locus. The kernels that were negative for YFP expression were the SSI null segregants.

By controlling the expression of ZmBBM and ZmWUS2 with developmental and inducible promoters, these developmental genes have been used to facilitate RMCE at numerous different target loci.

Example 6 Gene Targeting Using Homing Endonucleases

Molecular cloning and vector construction methods are well known and any such methods can be used to generate constructs to provide elements such as double-strand break-inducing enzymes, artificial target sites, targeting vectors, cell proliferation factors, or any other useful element. Vector construction is performed using standard molecular biology techniques. Any method of transformation can be used, and vector construction and/or insert preparation can be modified accordingly.

DNA double-strand break-inducing enzymes, such as an endonuclease, create double-strand breaks in the genome. Subsequent repair of the break can produce a mutation, DNA insertion, and homologous recombination products. In this manner, a double-strand break-inducing enzyme can be used for targeted modification of the genome to introduce a mutation, targeted insertion, or homologous recombination at a target locus. It is expected that the provision of one or more cell proliferation factors will enhance the targeted modification rates with double-strand break methods. Increased modification rates are expected at both artificial and endogenous target locus sites. Similarly, cell proliferation factors may also increase the rate of recovery of events in which a modification has occurred at the target locus. For example, one or more cell proliferation factors can be provided by introducing expression cassettes (e.g., Ubi Pro::Ubi intron::ZmBBM::pinII+nos Pro::ZmWUS2::pinII), resulting in enhanced gene targeting rates.

A. Artificial Target Site

An artificial target site (ATS) construct (ATS2) was constructed using a MDTP tetra-peptide linker to create a translational fusion between the selectable markers MoPAT (U.S. Pat. No. 6,096,947) and YFP(PHP21829). An in-frame insertion of the I-SceI recognition sequence in front of the MDTP-linker sequence of PHP21829 resulted in PHP22710. Upon delivery of the PHP21829 or PHP22710 construct into Hi-II maize immature embryos for functional evaluation, spots of yellow fluorescence were observed, confirming expression of the marker. Three stop codons were added to the PHP22710 fusion construct in front of the YFP coding sequence to create the artificial target site 2 (ATS2, PHP22709) construct. PHP22709 comprises the following operably linked components: Ubi pro::FLPm-rice actin pro::moPAT/I-SceI site/YFP::pin II-gAt. As expected, no visible yellow fluorescence was observed in Hi-II embryos bombarded with PHP22709.

ATS2 was designed with a minimal amount of sequences derived from maize to facilitate the interpretation of results. moPAT and YFP provide 5′ and 3′ homologous regions (˜1 kb and ˜4.1 kb, respectively) for targeting in homologous recombination experiments. Homology of the 3′ region was increased through the addition of 1578 bp of non-coding genomic sequence from Arabidopsis (gAt) following the pinII terminator. A FLP expression cassette was included in some experiments in order to test certain targeting vectors and other experimental design strategies.

B. Targeting Vectors

Several versions of targeting vectors were generated for delivery into maize embryos. Targeting vectors were designed that comprise a maize codon-modified I-SceI (moI-SceI) meganuclease expression vector derived from PHP22603 (U.S. Patent Application Publication No. 2009/0133152, which is herein incorporated by reference) and a positive selectable GAT4621 marker gene, flanked by two DNA segments homologous to the ATS2 target site. The homologous segments are 3019 bp (HR1) and 924 bp (HR2), respectively, in length. The GAT4621 gene is asymmetrically positioned within the homologous region to facilitate the identification of homologous recombinants by PCR. The basic vector was named TV-ATS2 (Targeting Vector for Artificial Target Site #2) and comprises the following operably linked components: Ubi pro::ubi 5′ UTR::mol-SceI::pinII-HR1-ubi pro::ubi 5′ UTR::GAT4621::pinII-HR2

A second targeting vector, named TV-ATS2Eraser, has two FRT sites directly flanking the TV-ATS2 elements, and was designed to provide a method to eliminate random integration events from selected material and to enrich the recovery of targeted events. TV-ATS2Eraser comprises the following operably linked components: FRT-ubi pro::ubi 5′UTR::mol-SceI::pinII-HR1-ubi pro::ubi 5′ UTR::GAT4621::pinII-HR2-FRT

A third targeting vector (TV-ATS2Turbo) carries a T-DNA replication cassette. Replicating T-DNAs are expected to persist longer in the transformed cells, providing more substrate and time for DNA recombination, including homologous recombination. Replication activity is provided by a modified version of the wheat dwarf virus replication-associated protein (Rep) lacking the intron sequences between the two open reading frames RepA and RepB, along with its cognate origin of replication (LIR). The replicase function of Rep is provided by the longer transcript encompassing two open reading frames (RepAB). Testing confirmed replication activity in BMS cells upon the delivery of the TV-ATS2Turbo cassette. It is possible that strong expression of RepAB may negatively impact the growth of transformed tissues. If this is the case, the Rep cassette may also act as a form of negative selection against random integrations, thus helping to identify potential target modification events. TV-ATS2Turbo comprises the following operably linked components: Ubi pro::ubi 5′ UTR::mol-SceI::pinII-WDV SIR::RepAB::WDV LIR-HR1-ubi pro::ubi 5′ UTR::GAT4621::pinII-HR2.

A fourth targeting vector, TV-ATS2TurboEraser, combines all the elements of the TV-ATS2Turbo vector, including the moI-SceI expression cassette, the GAT4621 marker for selection of all transformation events, the RepAB gene for amplification of T-DNAs, and FRT sites to reduce the number of randomly integrated T-DNAs in selected material. TV-ATS2TurboEraser comprises the following operably linked components: FRT-Ubi pro::ubi 5′ UTR::moI-SceI::pinII-WDV SIR::RepAB::WDV LIR-HR1-ubi pro::ubi 5′ UTR::GAT4621::pinII-HR2-FRT.

A fifth targeting vector (TV-PHP30662) was constructed using the same elements as TV-ATS2, but the vector lacks the regions of homology to the target site. TV-PHP30662 comprises the following operably linked components: Ubi pro::ubi 5′ UTR::moI-SceI::pinII-ubi pro::ubi 5′ UTR::GAT4621::pinII.

C. Maize Lines Comprising a Target Site

Maize lines comprising an artificial target site stably integrated into the genome were produced by Agrobacterium-mediated transformation. Zea mays Hi-II immature embryos were transformed using Agrobacterium-mediated transformation essentially as described in Djukanovic et al. (2006) Plant Biotech J 4:345-57. Briefly, 10-12 DAP immature embryos (1-1.5 mm in size) were dissected from sterilized kernels and placed into liquid medium. After embryo collection, the medium was replaced with 1 ml Agrobacterium (at a concentration of 0.35-0.45 OD550) containing a T-DNA comprising an artificial target site, e.g, ATS2 (PHP22709). Maize embryos were incubated with Agrobacterium for 5 minutes at room temperature, and then the mixture was poured onto a media plate. Embryos were incubated axis down, in the dark for 3 days at 20° C., then incubated 4 days in the dark at 28° C., followed by a transfer to new media plates containing 3.0 mg/L Bialaphos and 100 mg/L carbenicillin. Embryos were subcultured every three weeks until transgenic events were identified. Somatic embryogenesis was induced by transferring a small amount of tissue onto regeneration medium (containing 0.1 μM ABA, 1 mg/L IAA, 0.5 mg/L zeatin, 1.5 mg/L Bialaphos, and 100 mg/L carbenicillin) and incubated in the dark for two weeks at 28° C. All material with visible shoots and roots was transferred onto media containing 4.3 g/L MS salts (Gibco 11117), 5.0 ml/L MS Vitamins Stock Solution, 100 mg/L myo-inositol, 40.0 g/L sucrose, and 1.5 g/L Gelrite, pH 5.6, and incubated under artificial light at 28° C. One week later, plantlets were moved into glass tubes containing the same medium and grown until they were sampled and/or transplanted into soil.

Results

A total of 20 T0 transgenic plants were generated. Nineteen T0 plants survived to maturity. Leaf samples from these plants were collected for Southern analysis. Only single copy events that produced greater than 10 T1 kernels were used for further experiments. Twelve T0 events were identified from this process. T1 seeds produced by T1 self pollinations were planted for further characterization to confirm single copy ATS2 events by T1 segregation analysis. PAT activity was determined using a PAT protein detection kit. Four events (59, 60, 99, and 102) showed 1:2:1 Mendelian segregation for the target site. Events 99 and 102 also showed a 3:1 segregation of PAT expression, which also verified that the selected events were transcriptionally active. A total of 68 homozygous plants were produced from six selected single copy events and moved to the greenhouse for seed amplification and embryo production for transformation. Of the six selected events, events 59 and 99 showed a good tassel/ear developmental coordination. Embryos from these two events were used for a FLP activity assay to further confirm that the target site was transcriptionally active and to verify FLP function. FLP activity was assessed with the PHP10968 construct, in which the uidA coding sequence and the maize ubiquitin sequence is separated by the GFP coding sequence flanked by two FRT sites. FLP-mediated excision of this fragment is expected to reconstitute GUS expression. Every embryo from these events had GUS activity, indicating that ATS2 target sites in the two independent events were transcriptionally active. Six homozygous, single copy transgenic maize lines containing the ATS2 fragment were produced. Hemizygous embryos can be produced for re-transformation experiments by backcrossing or outcrossing. An ATS homozygous line is crossed to non-transgenic parental plants in order to produce the ATS hemizygous embryos for re-transformation experiments. All dissected embryos contained one copy of the artificial target site.

D. Target Site Modification

Agrobacterium-mediated transformation, as described elsewhere herein, is used to re-transform 9-12 DAP immature target line embryos comprising the ATS2 target site. The target line embryos are transformed with an I-SceI expression vector, and/or a targeting vector, with or without the following cassette: Ole Pro::ZmBBM::pinII+nos Pro::ZmWUS2::pinII+ALS Pro::Zm-ALS (HRA)::pinII Zm-ALS (HRA) is the maize acetolactase synthase with two mutated amino acids, making it resistant to sulfonylurea herbicides. Transgenic embryos containing the artificial target site (ATS2) are re-transformed with the targeting vectors delivered on T-DNA molecules. The target sites contain the I-SceI restriction site and the targeting vectors provide the I-SceI meganuclease activity. Re-transformation of transgenic embryos containing ATS2 with an I-SceI expression cassette produces double-strand breaks at the target site. As a result, targeted modifications including short deletions and other rearrangements are introduced at the target site. A GAT expression cassette is used to confirm construct delivery, therefore embryo co-cultivation is followed by callus selection on media containing 1 mM glyphosate. Transgenic callus events are resistant to glyphosate and exhibit blue fluorescence. In the re-transformation experiments for targeting, the selection protocol does not rely on activation/inactivation of moPAT::YFP; instead, all glyphosate-resistant, CFP+ events are screened by PCR for modifications of ATS indicative of targeting events.

For high-throughput PCR screening of large numbers of samples, DNA is extracted by a HotSHOT protocol (Truett et al. (2000) Biotechniques 29:53-54). Briefly, one leaf punch, or a sample of equivalent size, 400 μl of extraction buffer (25 mM NaOH, 0.2 mM EDTA), and two stainless steel beads are placed in each tube of a Mega titer rack. The samples are ground and extracted by shaking in a Genogrinder at 1650 rpm for 30-60 seconds, then incubating for 60-90 minutes at 95° C. The extracts are cooled to room temperature, 400 μl neutralization buffer (40 mM Tris-HCl, pH 5.0) is added, and the extracts are shaken at 500 rpm for 20-30 minutes. The samples are centrifuged at 4000 rpm for 5-10 minutes, followed by the collection of the supernatant. Two μl of the supernatant from each sample is used for PCR.

For further evaluation of putative transformation events, DNA extraction is performed using the Qiagen Dneasy Plant Mini kit according to the provided protocol (Qiagen Inc., Valencia, N. Mex., USA). PCR reactions contain 2 μl of DNA extract (100-200 ng), 10 μl of RedExtractandAmpPCR mix (R4775, Sigma, St. Louis, Mo.), 0.05 μl of each primer at a 100 μM concentration, and 7.9 μl water. The Expanded Long Template PCR amplification system (Roche Molecular Biochemicals, Indianapolis, Ind.) is used to amplify products of about 3 kb or larger. The Eppendorf Mastercycler Gradient cycler (Eppendorf North America, Westbury, N.Y.) is used with a PCR program specific for the particular primer annealing temperature and length of the desired PCR product. PCR products are evaluated and purified by agarose gel electrophoresis, by loading 15 μl of each PCR reaction on a 1% agarose gel. PCR products are purified using a Qiagen PCR purification kit (Qiagen Inc., Valencia, N. Mex.). Products less than 4 kb are directly sequenced, or cloned into the pCR4-TOPO vector (InVitrogen, Carlsbad, Calif., USA). Longer PCR products are first cloned into a vector and then sequenced.

Three PCR primer pairs are used to identify and characterize the transformation events: an ATS primer pair, an I-SceI primer pair, and an HR primer pair. Selected putative targeting events are further characterized by DNA sequencing using BigDye Terminator chemistry on an ABI 3700 capillary sequencing machine (Applied Biosystems, Foster City, Calif.). Each sequencing sample contains either 0.4-0.5 μg plasmid DNA or about 10 ng of the PCR product, and 6.4 pmole primer. Sequences are analyzed using the Sequencher program.

Selected events are further analyzed by Southern blots. Leaf tissue (about 1-2 grams fresh weight) is ground into a fine powder with liquid nitrogen. Twenty ml Puregene® Cell Lysis Solution is added to each sample and incubated 1 hour at 64° C., while shaking at 750 rpm. Samples are centrifuged 10 minutes at 4,000 rpm. DNA extract supernatants are transferred to new tubes, mixed with 5 ml of phenol/chloroform (1:1) solution, and centrifuged 10 minutes at 4000 rpm. The upper phase is removed, and mixed with an equal volume of isopropanol to precipitate the DNA. The solutions are centrifuged for 10 min at 4000 rpm, followed by removal of the supernatant and the resuspension of pellets in 5 ml of TE buffer, pH 8.0, 0.4 ml of ethidium bromide (10 mg/ml), and 5 g of cesium chloride. The mixture is centrifuged overnight (12-17 hrs) at 390,000 g. The DNA extraction and ethidium bromide removal are performed essentially as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY. The final DNA preparations are dissolved in TE buffer to yield 1.0 μg/μl DNA solutions. Ten μg DNA from each sample is digested overnight with 50 units of selected restriction enzyme(s) and the resultant digestion product(s) are separated on a 0.7% agarose gel run at 35 mV overnight. The TurboBlotter and Blotting Stack (Schleicher & Schuell, Keene, N.H.) are used to transfer DNA onto a nylon membrane as described in the manufacturer's manual. The DNA fragments are linked to the membrane by UV irradiation at 1.2 kjoules/m² in a UV Stratalinker (Stratagene, Cedar Creek, Tex.). The blots are pre-hybridized 2-3 hrs in 20 ml of ExpressHyb hybridization solution (Clontech, Palo Alto, Calif.) at 65° C. The random prime labeling system (Amersham Pharmacia Biotech, Piscataway, N.J.) is used with Redivue [³²P]dCTP to produce radioactively labeled DNA fragments according to the supplied protocol. Hybridizations are incubated overnight at 65° C. Blots are washed twice with 1% SSCE/0.1% SDS solution for 15 min at 65° C. and then two additional washes are done with 0.1% SSCE/0.1% SDS under the same conditions.

E. Homing Endonuclease Activity in Plant Cells

It is beneficial to be able to evaluate the relative DNA cleavage activity in plant cells of any native, modified, or custom-designed double-strand break inducing agent, for example a meganuclease or zinc-finger nuclease. Modifications include changes to meganuclease polynucleotide or amino acids sequences, such as codon optimization, UTRs, amino acid substitutions, or fusions. The meganuclease and target sequence can be provided to the plant cell using any appropriate delivery method. Any meganucleases and target sequences can be tested in any plant cells in this manner.

Briefly, a sequence encoding the homing endonuclease (EN) with its cognate target site sequence (TS) is integrated into a DNA construct, for example a T-DNA, and delivered to the plant cells. This construct also includes a recombinase, recombinase sites for excision, and viral replication elements. After a specified period of time, or at defined time points in a series, total DNA is extracted from the treated plant cells and used to transform E. coli. Only circular DNAs containing the target sites will be capable of transforming and propagating in E. coli. These DNA molecules are recovered from E. coli and at least a subset of these samples are analyzed for mutations produced by double-strand breaks at the target site. Mutated target sites can be identified by sequencing of PCR products, real-time PCR using fluorescent probes, PCR-based melting curve analysis, or other suitable methods.

For example, a T-DNA construct containing the following operably linked components is constructed: RB-FRT-cole1 ori-F1 ori-AMP-TS-WDV LIR-REP Exon1-REP Intron-REP Exon1-WDV SIR-FRT-UBI pro-UBI intron1-FLPm Exon1-ST LS1 Intron2-FLPm Exon2-pinII term-35S Enh-MN/ST LS Intron2-Ubi Intron1-Ubi Pro-LB-SPC-cole1 ori-COS. SPC is a bacterial gene conferring resistance to spectinomycin.

The coding regions for both the homing endonuclease (EN) and the recombinase (FLPm) contain an intron (e.g., ST-LS Intron 2) to suppress the expression of the proteins in bacterial cells (Agrobacterium or E. coli). This vector can be constructed using FLP-mediated recombination between a WDV replicase expression vector containing the target site sequence and an acceptor T-DNA vector containing FLP and the MN.

Agrobacterium containing a plasmid with the above components is used to transform BMS cells. In BMS cells, the meganuclease is expressed and can act upon the target site sequence. FLP recombinase is also expressed, excising the TS-containing WDV replicase expression vector, which circularizes and replicates. The acceptor T-DNA vector may also circularize, but cannot replicate. Replication amplifies the quantity of circular TS-containing WDV replicon, which will be the predominant DNA provided to E. coli. Six days after transformation, total DNA is isolated from the BMS cells and used to transform E. coli. E. coli colonies are screened sequentially for resistance to ampicillin and resistance to spectinomycin to identify colonies containing Ti plasmid DNA. Ampicillin-resistant colonies are selected and screened for mutations at the target site. The target sites can be recovered either by extraction of plasmid DNA from the E. coli, or by PCR amplification. PCR amplification reactions allow more efficient analysis of a large number of samples. Mutated target sites can be identified by sequencing of PCR products, real-time PCR using fluorescent probes, PCR-based melting curve analysis, or other suitable methods.

A summary of homing endonuclease and target site assay results are summarized in Table 3, wherein the I-SceI, I-CreI, Lig3-4, Lig3-4+, Lig3-4++homing endonucleases are combined with the corresponding target site (single or double copy).

TABLE 3 A summary of homing endonuclease and target site assay results. Homing # clones # Mutation Target Site endonuclease sequenced mutations rate I-SceI None 34 0  0% I-SceI I-SceI 58 49 84% Double I-SceI I-SceI 63 57 90% I-CreI None 34 0  0% I-CreI I-CreI 904 318 35% Double I-CreI I-CreI 66 50 76% LIG-1 Lig3-4 637 3 0.5%  LIG-1 Lig3-4+ 353 1 0.3%  LIG-1 Lig3-4++ 237 56 24%

Example 7 Targeted Modification of an Endogenous Genomic Locus

A genomic sequence near the liguleless1 locus on chromosome 2 was characterized for use as an endogenous targeting locus. The targeting construct comprised a UBL:moPAT::pinII expression cassette flanked by 3150 bp and 1255 bp of sequence homologous to that of the endogenous genomic locus, in addition to a UBI PRO::I-CRE SC (LIG3/4)::pinII expression cassette encoding a homing endonuclease specific for the endogenous sequence ATATACCTCACACGTACGCGTA (SEQ ID NO: 56).

The targeting plasmid was delivered at 100 ng plasmid/bombardment to scutellar cells of PHWWE immature embryos either alone, or with 25 ng each of PHP21875 (UBI::ZmBBM::pinII) and PHP21139 (In2-2 PRO::ZmWUS2::In2-1 TERM). After particle bombardment of 569 embryos with all three plasmids, 74 callus events were selected for resistance to bialaphos, and one of these events produced a positive band after PCR screening across the newly formed hybrid junction identifying a putative homologous recombination event. All eight plants regenerated from this event produced a positive PCR signal. Long range PCR, producing longer bands across the newly formed junctions were then used to further confirm successful introduction of the UBI::moPAT::pinII fragment into the endogenous LIG locus. Subsequent Southern analysis demonstrated that after cutting genomic DNA with either PstI or BamHI for probing with Probe 1, or cutting with SpeI or DraI for probing with Probe 2, the expected band sizes were observed which were indicative of perfect integration. Finally, PCR was used to verify that moPAT had integrated as a single copy, and that the I CREI (LIG), ODP2 and WUS2 transgenic expression cassettes had not integrated into the genome. To date, two homologous recombination events have been identified and verified when ODP2 and WUS2 were co-delivered with the donor plasmid, after analyzing approximately 310 events to recover the first perfect homologous recombination (HR) and 74 events to recover the second perfect HR. In separate testing without ODP2 and WUS2, approximately 280 transgenic events were analyzed and no perfect homologous recombination events have been recovered.

Additionally, the developmental genes ZmBBM and ZmWUS2 have also been used to facilitate integration of transgenes at two different endogenous target sites on chromosome 1.

Example 8 Identification of BBM Motifs

Fifty genes from different plant species were identified through a homology search using the maize BBM amino acid sequence (SEQ ID NO: 2) queried against annotated protein sequences (see FIG. 1). The gene structure and sequences of these BBM homologs were manually inspected and compared with EST/cDNA alignments whenever possible. The fifty polypeptides are set forth in SEQ ID NOs: 2, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 61-96. To systematically identify possible motifs within the BBM homologs, protein sequences of these fifty homologs were submitted to the MEME web server, available on the world wide web at meme.nbcr.net/meme4_(—)1/cgi-bin/meme.cgi, with the following specific parameters:

Number of different motifs: 20

Minimum motif width: 5

Maximum motif width: 300

Minimum number of sites: 5

Default values were applied for all other parameters. The raw results from MEME were manually compared with multiple sequence alignments generated by clustalw. Only those candidates showing good consensus with the sequence alignments were considered as motifs for further analysis.

The fifty genes were subjected to a phylogenetic analysis and a total of six subgroups were identified, including BBM, PLT3, PLT1/2, AIL6/7, AIL1, and ANT (see FIG. 1). FIG. 3 depicts all 50 sequences with each of the motifs that were identified using the MEME web server. FIG. 2 provides the motif consensus sequences along with alignments of the various polypeptides used by the MEME web server to generate the consensus motif. With a few exceptions, motifs 1-6, as defined immediately hereinbelow, are present in all 50 genes. This includes motifs 1-3 (SEQ ID NOs 3-5, respectively), which represent the two AP2 domains and a sequence linking the two domains (linker sequence). Motif 4, with the consensus sequence of PK[L/V][E/A][D/N]FLG (SEQ ID NO: 6) is amino-terminal to the two AP2 domains. Motif 5 (SEQ ID NO: 7) flanks the two AP2 domains on the carboxy terminal end of the polypeptides. Near the amino terminus of the polypeptides is motif 6, with the consensus sequence of NWL[G/S]FSLSP (SEQ ID NO: 8).

There were motifs that were relatively specific for the BBM subgroup of the homologous sequences (referred to herein as BBM polypeptides). An alignment of the BBM polypeptides can be found in FIG. 4. Motif 7 is found in all BBM polypeptides at the amino terminus of the polypeptide and has the consensus sequence of [G/E]LSMIK[T/N]WLR (SEQ ID NO: 9). Another motif that is present in all of the BBM polypeptides except for the polypeptides from Brassica and from Arabidopsis is Motif 10. Motif 10 has the consensus sequence of WCK[Q/P]EQD (SEQ ID NO: 12) and is located downstream of the AP2 domains.

There are three more motifs specific to the BBM group of polypeptides, including Motif 15 (SEQ ID NO: 14) which appears only in BBM orthologs, but not in the monocot BBM2 polypeptides; a monocot specific motif (Motif 19; SEQ ID NO: 15); and a general BBM specific motif (Motif 14; SEQ ID NO: 13), which appears in BBM homologs except for the Brassica and legume branch.

FIG. 5 provides a summary of the motif structure of the BBM homologs. The amino terminal motifs 4 and 6 and the AP2 flanking motif 5 distinguish the BBM homologous sequences from other two AP2 domain-containing homologs, such as WRI, AP2, and RAP2.7. Therefore, motifs 1-6 can be considered as core BBM/PLT family motifs. Many subgroups of the BBM/PLT family (BBM, PLT1/2, AIL1, and ANT) also have a carboxy-terminal motif (motif 8; SEQ ID NO: 10) and the third amino terminal motif (motif 9; SEQ ID NO: 11).

The BBM polypeptides all have one additional motif (motif 7; SEQ ID NO: 9) in the amino terminus, and all but the Brassica and Arabidopsis BBM homologs have an AP2 downstream motif (motif 10; SEQ ID NO: 12). Some other BBM/PLT family members (e.g., monocot AIL1) may have a similar motif as motif 7, but none of them also have motif 9. Motif 10 appears only in BBM polypeptides. In summary, the MEME predicted motifs 1-10 can be regarded as BBM polypeptide motifs. All monocot BBM polypeptides (corn, sorghum, and rice) also have motif 14, 15, and 19 (see FIG. 3). Some dicot BBM polypeptides and the second monocot BBM group (BBM2) have one or two of these motifs, but none have all three motifs.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. 

That which is claimed:
 1. A method for modifying a target site of a plant cell, wherein said target site of said plant cell comprises a recognition sequence, and wherein said method comprises: a) introducing into said plant cell at least one heterologous polynucleotide encoding the polypeptide of SEQ ID NO: 2, and expressing said heterologous polynucleotide; and b) introducing a heterologous polynucleotide encoding a double-strand break-inducing enzyme and expressing said heterologous polynucleotide encoding said double-strand break-inducing enzyme, wherein said double-strand break-inducing enzyme recognizes said recognition sequence and introduces a double-strand break at or near the recognition sequence to produce a modified target site.
 2. The method of claim 1, wherein said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2, is operably linked to a promoter active in said plant.
 3. The method of claim 2, wherein said promoter operably linked to said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2, is an oleosin promoter, a ubiquitin promoter, a nopaline synthase promoter, or a In2 promoter.
 4. The method of claim 1, wherein said modified target site comprises a deletion, a mutation, a replacement, or an integration of a nucleotide sequence when compared to said target site.
 5. The method of claim 1, wherein said double-strand break-inducing enzyme is selected from the group consisting of an endonuclease, a zinc finger nuclease, a transposase, a topoisomerase, and a site-specific recombinase.
 6. The method of claim 5, wherein said endonuclease comprises a homing endonuclease.
 7. The method of claim 6, wherein said homing endonuclease comprises a modified endonuclease that has been modified to specifically bind, said recognition sequence.
 8. The method of claim 7, wherein said modified homing endonuclease is derived from a homing endonuclease selected from the group consisting of I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ceu-1, 1-CeuAIIP, I-Cre1, 1-CrepsbIP, I-Crepsb11P, 1-Crepsb111P, 1-Crepsb1VP, I-Tli1, I-Ppo1, PI-Psp1, F-SceI, F-SceII, F-Suv1, F-Tev1, F-Tev11, I-Ama1, I-Ani1, I-Chu1, I-Cmoe1, I-Cpa1, I-Cpa11, I-Csm1, I-Cvu1, 1-CvuAIP, I-Ddi1, I-Ddi11, I-Dir1, I-Dmo1, I-Hmu1, I-Hmu11, I-HsNIP, I-Lla1, I-Mso1, I-Naa1, I-Nan-1, 1-Ncl1P, 1-Ngr1P, I-Nit1, I-Nja1, I-Nsp2361P, I-Pak1, 1-Pbo1P, 1-Pcu1P, 1-PcuAI, 1-PcuVI, 1-Pgr1P, 1-Pob1P, I-Por-1, 1-Por11P, 1-Pbp1P, 1-SpBeta1P, I-Sca1, 1-Sex1P, 1-Sne1P, I-Spom1, I-SpomCP, 1-Spom1P, 1-Spom11P, 1-Squ1P, 1-Ssp68031, 1-SthPhiJP, I-SthPhiST3P, 1-SthPhiSTe3bP, 1-Tde1P, I-Tev1, I-Tev11, I-Tev111, 1-UarAP, I-UarHGPAIP, 1-UarHGPA13P, 1-Vin1P, 1-Zbi1P, PI-Mtu1, PI-MtuHIP P1-MtuHIIP, P1-Pfu1, P1-Pfu11, P1-Pko1, P1-Pko11, PI-Rma43812IP, PI-SpBeta1P, P1-Sce1, P1-Tfu1, P1-Tfu11, P1-Thy1, PI-TIiI, and PI-TiiII.
 9. The method of claim 1, wherein said double-strand break-inducing enzyme is a site-specific recombinase and said recognition sequence comprises a first recombination site.
 10. The method of claim 9, wherein said site-specific recombinase is selected from the group consisting of FLP, Cre, SSV1, R, Gin, lambda Int, phiC31 Int, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, U153, and HK022 Int.
 11. The method of claim 9, wherein said target site further comprises a second recombination site, wherein said target site comprises the following operably linked components: said first recombination site, a nucleic acid sequence, and a second recombination site.
 12. The method of claim 11, wherein said first recombination site is recombinogenic with the second recombination site in the presence of said site-specific recombinase.
 13. The method of claim 12, wherein said nucleic acid sequence is excised or inverted to produce the modified target site.
 14. The method of claim 1, wherein said modified target site comprises an integrated polynucleotide of interest, and wherein said method further comprises introducing into said plant cell a transfer cassette comprising said polynucleotide of interest.
 15. The method of claim 14, wherein said transfer cassette comprises at least a first region having homology to said target site.
 16. The method of claim 15, wherein said transfer cassette comprises in the following order: said first region of homology to said target site, said polynucleotide of interest, and a second region of homology to said target site.
 17. The method of claim 1, said method further comprising identifying cells comprising the modified target site and regenerating a plant having the modified target site.
 18. The method of claim 17, wherein said method further comprises reducing the activity of said polypeptide of SEQ ID NO: 2 prior to regenerating a plant having the modified target site.
 19. The method of claim 18, wherein reducing the activity of said polypeptide of SEQ ID NO: 2 comprises excising said heterologous polynucleotide encoding said polypeptide of SEQ ID NO:
 2. 20. The method of claim 19, wherein said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 is flanked by recombination sites, and wherein said method further comprises introducing into said plant cell a site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2, whereby said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 is excised in the presence of said site-specific recombinase.
 21. The method of claim 20, wherein said introducing said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 comprises introducing a heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 and expressing said heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said polypeptide of SEQ ID NO:
 2. 22. The method of claim 21, wherein said heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 is operably linked to an inducible promoter.
 23. The method of claim 21, wherein said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 and said heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 are flanked by said recombination sites, whereby said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 and said heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 is excised in the presence of said site-specific recombinase.
 24. The method of claim 23, wherein said plant cell further comprises a heterologous polynucleotide encoding a Wuschel polypeptide, and wherein said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2, said heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2, and said heterologous polynucleotide encoding said Wuschel polypeptide are flanked by said recombination sites, whereby said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2, said heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2, and said heterologous polynucleotide encoding said Wuschel polypeptide is excised in the presence of said site-specific recombinase.
 25. The method of claim 24, wherein said heterologous polynucleotide encoding said Wuschel polypeptide is operably linked to a nopaline synthase promoter or an In2-2 promoter.
 26. The method of claim 24, wherein said heterologous polynucleotide encoding said Wuschel polypeptide is stably integrated into the genome of said plant cell.
 27. The method of claim 24, wherein said heterologous polynucleotide encoding said Wuschel polypeptide is transiently expressed.
 28. The method of claim 24, wherein said heterologous polynucleotide encoding said Wuschel polypeptide has a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO: 51, 57, 99, or 97; b) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 51, 57, 99, or 97; c) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 52, 58, 100, or 98; and d) a nucleotide sequence encoding an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 52, 58, 100, or
 98. 29. The method of claim 24, wherein said method further comprises reducing the activity of said Wuschel polypeptide prior to the regeneration of a plant having the modified target site.
 30. The method of claim 1, wherein said plant cell is a dicot plant cell.
 31. The method of claim 1, wherein said plant cell is a monocot plant cell.
 32. The method of claim 31, wherein said monocot plant is selected from the group consisting of maize, rice, sorghum, barley, wheat, millet, oats, sugarcane, turfgrass, and switch grass.
 33. A method for targeting the insertion of a polynucleotide of interest to a target site in a plant cell, wherein said target site comprises a first recombination site, said method comprising: a) introducing into said plant cell at least one heterologous polynucleotide encoding a polypeptide of SEQ ID NO: 2 and expressing said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2; b) introducing into said plant cell a transfer cassette comprising a second recombination site and said polynucleotide of interest, wherein the first and said second recombination sites are recombinogenic with respect to one another; and c) introducing into said plant cell a site-specific recombinase that recognizes and implements recombination at said first and said second recombination sites, thereby inserting said polynucleotide of interest at the target site.
 34. A method for targeting the insertion of a polynucleotide of interest to a target site in a plant cell, wherein said target site comprises a first and a second recombination site, wherein said first and said second recombination sites flank a nucleotide sequence and are non-recombinogenic with respect to one another, said method comprising: a) introducing into said plant cell at least one heterologous polynucleotide encoding the polypeptide of SEQ ID NO: 2 and expressing said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2; b) introducing into said plant cell a transfer cassette comprising a third and a fourth recombination site flanking said polynucleotide of interest, wherein the third recombination site is recombinogenic with the first recombination site, and wherein the fourth recombination site is recombinogenic with the second recombination site; and c) introducing into said plant cell a site-specific recombinase that recognizes and implements recombination at the first, second, third, and fourth recombination sites; thereby replacing the nucleic acid sequence of the target site with the polynucleotide of interest from the transfer cassette.
 35. A method to integrate multiple transfer cassettes at a target site in a plant cell, wherein said target site comprises at least a first and a second recombination site, said method comprising: a) introducing into said plant cell a first transfer cassette comprising in the following order: at least the first, a third, and the second recombination sites, wherein the first and the third recombination sites of the first transfer cassette flank a first polynucleotide of interest, and wherein said first, said second, and said third recombination sites are non-recombinogenic with respect to one another; b) introducing into said plant cell a first site-specific recombinase, wherein said site-specific recombinase recognizes and implements recombination at the first and the second recombination sites; c) introducing a second transfer cassette comprising at least the second and the third recombination sites, wherein the second and the third recombination sites of the second transfer cassette flank a second polynucleotide of interest; and d) introducing into said plant cell a second site-specific recombinase, wherein said second site-specific recombinase recognizes and implements recombination at the second and third recombination sites; whereby the first and the second transfer cassettes are integrated at the target site of the plant cell, and wherein said method further comprises introducing at least one heterologous polynucleotide encoding the polypeptide of SEQ ID NO:2 into said plant cell and expressing said heterologous polynucleotide encoding said polypeptide before or during the introduction of the first site-specific recombinase, the second site-specific recombinase, or both the first and the second site-specific recombinase.
 36. A method to integrate multiple transfer cassettes at a target site in a plant cell, wherein said target site comprises in the following order at least a first, a second, and a third recombination site, wherein said first, said second, and said third recombination sites are non-recombinogenic with respect to one another, said method comprising: a) introducing into said plant cell a first transfer cassette comprising a first polynucleotide of interest flanked by the first and the second recombination sites; b) introducing into said plant cell a first site-specific recombinase, wherein said first site-specific recombinase recognizes and implements recombination at the first and the second recombination sites; c) introducing a second transfer cassette comprising a second polynucleotide of interest flanked by at least the second and the third recombination sites; and d) introducing into said plant cell a second site-specific recombinase, wherein said second site-specific recombinase recognizes and implements recombination at the second and third recombination sites; whereby the first and the second transfer cassettes are integrated at the target site of the plant cell, and wherein said method further comprises introducing at least one heterologous polynucleotide encoding the polypeptide of SEQ ID NO: 2 into said plant cell and expressing said heterologous polynucleotide encoding said polypeptide of SEQ ID NO: 2 before or during the introduction of the first site-specific recombinase, the second site-specific recombinase, or both the first and the second site-specific recombinase.
 37. A plant cell comprising a target site, wherein said target site of said plant cell comprises a recognition sequence, and wherein said plant cell further comprises at least one heterologous polynucleotide encoding the polypeptide of SEQ ID NO: 2 operably linked to a promoter active in said plant cell, a double-strand break-inducing enzyme capable of recognizing said recognition sequence and introducing a double-strand break at or near the recognition sequence, and a transfer cassette comprising a polynucleotide of interest, wherein said transfer cassette comprises a first region of homology with said target site. 